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Article

Microbial Dynamics and Volatile Compound Profiles in Artisanal Kefir During Storage

by
Jeferson Aloísio Ströher
1,
Wemerson de Castro Oliveira
2,*,
Anderson Santos de Freitas
3,
Marcela Mendes Salazar
4,
Simone Hickmann Flôres
1 and
Patrícia da Silva Malheiros
1,*
1
Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Porto Alegre 90010-150, Brazil
2
REQUIMTE/LAQV, Department of Teaching, Research, and Extension, Federal Institute of Education, Science and Technology Sul-Rio-Grandense, Lajeado 95900-000, Brazil
3
Center for Nuclear Energy in Agriculture, University of São Paulo, Piracicaba 13416-000, Brazil
4
Laboratory of Genomics and Expression, State University of Campinas, São Paulo 13083-865, Brazil
*
Authors to whom correspondence should be addressed.
Fermentation 2025, 11(2), 105; https://doi.org/10.3390/fermentation11020105
Submission received: 20 January 2025 / Revised: 7 February 2025 / Accepted: 12 February 2025 / Published: 19 February 2025
(This article belongs to the Special Issue Dairy Fermentation, 3rd Edition)
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Abstract

:
Artisanal kefir is produced by fermenting milk with kefir grains, resulting in a lightly carbonated drink with health-promoting bioactive compounds. However, sensory variability and fermentation conditions challenge its standardization, limiting commercialization in Brazil due to regulatory requirements. This study evaluated the physicochemical properties, volatile compounds, and microbiological stability of artisanal kefir produced in southern Brazil during 30 days of storage. Metabarcoding analysis, carried out by sequencing the V3/V4 regions of the 16S rRNA gene (bacteria) and the ITS region (fungi), revealed an increase in bacterial diversity, with a predominance of Enterococcus and Acetobacter, while fungal diversity decreased, with a predominance of Kazachstania. The physicochemical parameters remained stable. The concentration of volatile compounds, analyzed using a gas chromatograph coupled to a mass spectrometer, decreased, except for an increase in 2-heptanol. The aromatic profile was enriched with alcohols and ketones, possibly influenced by Enterococcus and Acetobacter. These findings show that kefir maintained microbiological stability and adequate sensory characteristics throughout the period analyzed. The study provides subsidies for the standardization of artisanal kefir and compliance with Brazilian quality standards, as well as guiding future research into durability, quality, and consumer perception.

1. Introduction

Kefir is a traditional fermented milk product from the Caucasus, Mongolia, and Eastern Europe, widely appreciated for its sensory characteristics and potential health benefits [1,2]. With its creamy consistency and acidic-alcoholic taste, its popularity is growing in Western countries, where it is recognized as a functional food due to its antimicrobial, anti-inflammatory, and antioxidant properties, helping to prevent tumors and diabetes [3,4,5]. The term “kefir”, which, in Turkish, means “good feeling”, reflects the refreshing nature of the drink and its beneficial effects on health [6].
Artisanal kefir is produced by fermenting milk with kefir grains, which are complex consortia of bacteria and yeasts in a matrix of polysaccharides, proteins, and lipids [7]. This biological process converts milk into a slightly carbonated and acidic drink, rich in microbial metabolites such as organic acids, amino acids, ethanol, bioactive peptides, bacteriocins, vitamins, and exopolysaccharides, giving it a unique flavor and functional properties [1,8].
The production of artisanal kefir derives from kefir grains, composed of lactic acid bacteria (LAB), acetic acid bacteria (AAB), yeasts, and other fungal species, and is central to the final quality of the product. The predominant genera found in the literature include Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, and Acetobacter, as well as yeasts such as Saccharomyces and Kluyveromyces. The symbiotic interaction between these microorganisms during fermentation generates volatile compounds that characterize the aroma and taste of kefir [9,10,11].
Historically, kefir fermentation was an essential strategy for preserving milk in communities with limited access to modern preservation methods. However, large-scale production faces obstacles due to the variability in taste, aroma, and acidity caused by microbial interactions and fermentation conditions (time, temperature, and hygiene), making it difficult to standardize artisanal kefir [12]. Artisanal kefir is mostly acquired through the donating grains, which allows it to be propagated and produced at home. Few companies invest in its commercialization, limiting sales to local and home markets, with an estimated shelf life of between 5 and 7 days [13].
However, successive transfers, and variations in storage methods and handling without strict hygiene practices can compromise the microbiological quality of the product, resulting in significant variations in the composition and standardization of artisanal kefir [13]. In this context, the development of a standardized, safe, and easy-to-handle kefir is essential in order to meet the growing demand for functional foods and guarantee the stability of its beneficial properties [14]. In Brazil, the commercialization of artisanal kefir on an industrial scale there probably is not due to the difficulty in standardizing its microbiological and physicochemical profile, affecting its shelf life and its compliance with the Technical Regulation on Identity and Quality (RTIQ) for dairy products [15]. This regulation requires a maximum acidity of 1.0 g of lactic acid per 100 g and an alcohol content between 0.5% and 1.5% (v/v), and a minimum count of 10⁷ CFU/g of lactic acid bacteria and 10⁴ CFU/g of specific yeasts.
The sensory acceptance of kefir is strongly linked to factors such as acidity and flavor, which influence its appreciation among different consumer groups. In a study conducted in southern Brazil, participants in a local group expressed different preferences, with some highlighting the intense acidity and others comparing it to natural yogurt for its liquid texture and white appearance [16]. The increase in acidity, an attribute resulting from microbial growth during fermentation, is identified as one of the factors that demand standardization for the industrialization of kefir [17,18].
In recent decades, metabarcoding analysis has become an essential tool for characterizing the microbial communities in kefir grains and beverages. This approach allows for the precise identification of species and strains, overcoming the limitations of traditional culture-based methods [19,20]. Advances in sequencing technologies have been crucial for the detection of dominant microorganisms and minority populations with relevant ecological functions in kefir [21]. These discoveries not only broaden applications in health and the food industry but also drive innovation and the marketing of a more standardized artisanal kefir, allowing greater control over its microflora and microbiological quality [22,23].
In this study, we analyzed the physicochemical and microbiological composition of artisanal kefir samples in southern Brazil, with the aim of understanding the dynamics of microorganisms throughout the beverage’s shelf-life process, comparing the results with Brazilian regulatory requirements. Our hypothesis is that maturation leads to a reduction in microbial diversity, driven by an increase in acidity and a decrease in available nutrients. Furthermore, we postulate that the quality and attributes of the product would remain stable for a period of thirty days, ensuring suitable conditions for its safe marketing [16,24].

2. Materials and Methods

2.1. Raw Material and Kefir Production

The kefir grains were obtained from the ProBios Kefir e Saúde agroindustry (29°26′57′′ S 51°56′37′′ W), incubated at Tecnovates, in the Vale do Taquari Science and Technology Park, at the University of Vale do Taquari-RS (Lajeado, RS, Brazil), with support from the TechFuturo project of the Rio Grande do Sul State Department of Innovation, Science and Technology (SICT). The kefir batches used in the experiment were produced and analyzed throughout 2024, according to the flowchart in Figure 1.
Kefir was produced by adding 100 g of kefir grains to a glass jar with a lid, containing 2 L of whole ultra-high-temperature (UAT) milk. The jar was incubated in a B.O.D. incubator (model NT 705) under controlled conditions, maintaining a temperature of 25 ± 0.5 °C for a period of 18 h. After the fermentation process, the pH of the kefir was determined in five different places in the bottle using a portable pH meter.
Subsequently, the kefir was strained to separate the grains from the liquid, which were stored appropriately. The fermented beverage was homogenized and the pH was measured again, before being transferred to a new glass jar, where it was left to mature for an interval of 18 to 24 h in a controlled environment (25 ± 0.5 °C). After the maturation period, the kefir was homogenized again and the pH was checked. The final product was then filled manually into previously sterilized glass bottles. The bottles were dated, identified with the batch number, and stored in a refrigerator at a temperature below 8 °C, subjected to analysis throughout their shelf life [25].
Samples were taken after initial maturation (48 h) (0 days), and then at 15- and 30-day intervals in order to identify a safe shelf life for kefir. The physicochemical analyses were carried out in triplicate, while the analyses of volatile compounds and metabarcoding were carried out in duplicate, with the mean and standard deviation calculated and the appropriate statistical tests applied. The shelf life of the experiment was defined based on the average period in which the quality attributes, including the aromatic characteristics of artisanal kefir, remain stable. Furthermore, this period complies with Brazilian legislation for fermented milks [15,16,24,26].

2.2. Metabarcoding Analysis

The bacterial communities were identified using high-performance sequencing of the V3/V4 regions of the 16S rRNA gene. The libraries were prepared following the Neoprospecta Microbiome Technologies protocol, with amplification using specific primers for the V3–V4 region of the 16S rDNA gene: 341f and 806r (GGACTACHVGGGTWTCTAAT) [27]. To identify the fungal communities in the kefir samples, the ITS region was amplified using the ITS1F/ITS2R primers [28,29,30]. The libraries were sequenced on the MiSeq Sequencing System (Illumina Inc., San Diego, CA, USA), with 600-cycle V3 or 500-cycle V2 kits for paired-end sequencing.
For the analysis of microbial communities, raw reads were analyzed using the DADA2 pipeline [31], considering acceptable sequences with a quality score greater than 30. The filtered reads were grouped into amplicon sequence variants (ASVs) and associated with taxonomy using the SILVA database [32]. The resulting ASV table was imported into a phyloseq object [33] for subsequent analysis.

2.3. Analysis of Volatile Compounds

The analyses of volatile organic compounds (VOCs) were performed at the Technological Center for Food Research and Production (CTPPA) of Tecnovates (Lajeado, Rio Grande do Sul, Brazil), according to Bernardi et al. (2014) [34], using a gas chromatograph coupled to a mass spectrometer (GC-MS), model GCMS-QP 2010 Ultra (Shimadzu Corporation, Kyoto, Japan), equipped with a DB-5MS capillary column (30 m × 0.25 mm, 0.25 µm—Agilent, Santa Clara, CA, USA), for separation of volatile compounds. The oven-heating program was started at 40 °C, started for 3 min, followed by a heating ramp of 3 °C/min up to 150 °C, and, subsequently, by a ramp of 15 °C/min until reaching 300 °C, where it appeared for 2 min, totaling a chromatographic separation time of 51 min. Operating conditions included an injection temperature of 250 °C, interface temperature of 280 °C, ion source temperature of 230 °C, scan range of 40–600 m/z, electron impact ionization source (70 eV), helium attractor gas, column flow rate of 0.98 mL/min, column pressure of 43.1 kPa, and injection in *split* mode with a ratio of 5:1.
For sample preparation, 5 g of kefir was weighed into 20 mL headspace vials, which were sealed with silicone septum caps. The samples were heated to 60 °C for compound volatilization, and an SPME fiber (75 µm CAR/PDMS, 23 GA, Supelco, Bellefonte, PA, USA) was inserted into the vial and exposed for 40 min at the same temperature. After this period, the fiber was detailed to the injection compartment of the chromatograph, where it was heated to 250 °C for 2 min for desorption of the compounds, followed by cleaning at 250 °C for 15 min. The identification of the compounds was performed by comparing the fragmentation profiles obtained with the National Institute of Standards and Technology (NIST) spectral library.

2.4. Physico-Chemical Analysis of Kefir

2.4.1. pH Analysis

The pH analyses were carried out on the premises of the PróBios agro-industry (Lajeado, Rio Grande do Sul, Brazil), according to the Lutz method (2008), using a Marte digital pH meter (model MB-10).

2.4.2. Lactic Acidity Analysis

The determination of lactic acidity followed Brasil (2006) [35]: 10 g of the sample were weighed into a 250 mL Erlenmeyer flask, added to 10 mL of water at 20 °C and four drops of phenolphthalein, titrated with 0.1 N sodium hydroxide solution until a persistent pink color appeared, and converted into lactic acidity.

2.4.3. Lactose Analysis

Lactose was analyzed using the DNS method [36], modified [37].

2.4.4. Ethanol Analysis

The ethanol content was quantified in an external laboratory accredited by MAPA (SENAI Institute of Food and Beverage Technology, Chapecó-SC), in accordance with Normative Instruction no. 24 of 8 September 2005 [38], expressed as % (v/v).

2.4.5. Fat Analysis

Fat was determined using the modified Mojonnier method (AOAC 2012, no. 989.05).

2.4.6. Ash Analysis

For the ash analysis, the crucibles were first calcined in a muffle furnace at 550 °C for two hours and cooled for 30 min in a desiccator. Approximately 2 g of the sample was weighed, pre-evaporated, and heated at 250 °C for one hour, then heated to 550 °C to obtain white ash. After cooling, the mass was weighed again and the percentage of ash calculated.

2.4.7. Protein Analysis

Protein analysis followed Granjeiro et al. (2022) [39], with the preparation of a bovine serum albumin (BSA) standard curve and incubation of the samples with Bradford reagent; the absorbance at 595 nm was measured to calculate the protein concentration using the calibration curve equation.

2.4.8. Moisture Analysis

Moisture analysis was based on Castanheira (2012) [40]. The porcelain crucibles were heated to 102 ± 2 °C for one hour, cooled in a desiccator, and weighed. Then, 3 to 5 g of kefir were added to the crucibles and dried at 102 ± 2 °C for five hours. After cooling, the mass loss was calculated to determine the total solids content.

2.4.9. Color Analysis

Color analysis was carried out on a Konica Minolta Cr400 colorimeter using the CIELAB system (1976). Color measurements were performed using a D65 illuminant and a 10° observer, recording the L*, a*, and b* values, where L* represents the brightness of the color, ranging from 0 (black) to 100 (white), a* indicates the variation between green (−a) and red (+a), and b* represents the variation between blue (−b) and yellow (+b).

2.4.10. Total Solids Analysis

Total solids were quantified on an i-Thermo 163L moisture balance, and dried to constant weight, to calculate the residual mass.

2.5. Statistical Analysis

All bioinformatic and statistical analyses were carried out in the R environment (version 4.3.0) using the RStudio software (version 2023.09.1+494) [41]. The code for the analyses carried out in this study can be found publicly on GitHub at https://github.com/FreitasAndy/PSFforAmazonianPastures, accessed on 1 February 2025. The figures were produced using the ggplot2 package [42], and some of these figures were edited for aesthetic purposes only (i.e., changing colors and fonts) using the Inkscape 1.3.2 program. As the data did not fit the normal distribution, we proceeded with an analysis suitable for non-parametric data. We used the Kruskal–Wallis test followed by Dunn’s post hoc test [43] to test for differences unrelated to the microbiota.
Raw sequencing reads were analyzed using the DADA2 pipeline [31], considering acceptable sequences with an average quality score greater than 30. The filtered reads were grouped into amplicon sequence variants (ASVs) and associated with taxonomy using the SILVA v. 138.1 database [32]. The resulting ASV table was imported into both a phyloseq (X)] object and an R6 object from the microeco package [8], for subsequent analysis.
Alpha diversity was calculated considering the number of different taxa identified in each sample (observed diversity), and the dominance of taxa was calculated using the inverse Simpson’s index, considering a 95% confidence level using the Kruskal–Wallis test followed by Dunn’s post hoc test. Beta diversity was calculated by transforming the dataset into a centralized logarithmic ratio (CLR), to reflect the compositional structure of the data. The data were ordered using Euclidean scaling, and the non-metric multidimensional scaling was plotted on the first two axes. Significance was calculated by permutational multivariate analysis of variance (PERMANOVA), at a significance level of 5% and 999 permutations, using the adonis function of the vegan package [44]. Finally, correlation analyses were carried out at the genus level using the Spearman method (p < 0.05) in the corrplot library.

3. Results and Discussion

3.1. Microbial Distribution of Kefir Throughout Its Shelf Life

The microbiome of the kefir analyzed was dominated mostly by bacteria from the Enterococcus and Acetobacter genera, with the Acetobacter abundance decreasing after 15 days of maturation and remaining low after 30 days. Although little studied, like other LAB in artisanal kefir, Acetobacter and Enterococcus contribute to its acidification, with Acetobacter oxidizing ethanol to acetic acid and Enterococcus metabolizing sugars to lactic acid, both of which inhibit pathogens. Acetobacter is favored by aerobic environments and temperatures of 25–30 °C, while Enterococcus is more tolerant of pH variations and can grow anaerobically. Acetobacter contributes to sensory properties, while Enterococcus improves microbiological safety, but can pose risks due to pathogenic strains. The predominance of both is influenced by artisanal practices and fermentation conditions [10,45,46,47,48,49]. Factors such as the temperature, humidity, and the kefir fermentation substrate, among others, can influence the growth of specific microorganisms in artisanal kefir [48,50].
On the other hand, there was a small proportional increase in Lactococcus throughout maturation (Figure 2a). Lactobacillus, Lentilactobacillus, and Leuconostoc maintained relatively stable populations, albeit at extremely low levels.
The literature on artisanal kefir microbiota generally points to the genera Lactobacillus, Lentilactobacillus, Lactococcus, Leuconostoc and Acetobacter as the most abundant, with Lactobacillus typically being dominant in the microbial composition [9,19,51]. The microbial diversity of kefir is often highlighted, with studies showing the presence of Lactobacillus, Lentilactobacillus, Lacticaseibacillus, and Acetobacter [51], while the spatial distribution of microorganisms in kefir grains, with Lactobacillus predominating in the inner layers, also influences the final composition of the drink [9].
Variations in the microbial composition of artisanal kefir, produced on a small scale with traditional grains, reflect regional practices and directly influence the taste, texture, and nutritional profile of the product [11,25]. This regionalized context affects not only the probiotic potential of kefir, but also its health benefits, highlighting the complex relationship between production methods and the functionality of artisanal kefir.
Among the fungi identified, the genus Kazachstania was the most abundant on the first day, but its quantity decreased substantially after 30 days, a result in line with previous observations [2,52,53]. In contrast, Penicillium increased in abundance towards the end of this period, as did Aspergillus, which showed considerable growth over the 30 days. The Debaryomyces genus, which was initially abundant, peaked at 15 days, but decreased towards the end of the experiment. The genera Candida, Cladosporium, Meyerozyma, Papiliotrema, Rhizopus, and Simplicillium remained low in abundance, with minimal variations over time.
The decrease in fungal abundance in artisanal kefir can compromise its sensory characteristics, resulting in a reduction in flavor and aroma complexity. At the same time, the increase in lactic acid bacteria contributes to a greater acidity and a loss of effervescence CO₂ production by yeasts. It is, therefore, essential that we maintain a balance between the bacterial and fungal communities in order to preserve the product’s distinctive sensory properties [47,54,55,56].
Recent studies suggest an increase in the fungal diversity of kefir with fermentation time. For example, Brasiel et al. (2021) [57] observed an increase in the diversity of the fungal community after 30 days, highlighting Aspergillus sp., Cordyceps sp., and Saccharomyces sp. as the most prevalent genera. Similarly, Sumarmono et al. (2023) [58] identified Kazachstania and Kluyveromyces as the dominant genera in cow’s milk kefir in Indonesia, with small amounts of Aspergillus spp. and Botryotrichum. Although Aspergillus is less commonly reported in kefir, its ability to produce important enzymes, such as amylase, amyloglucosidase, and maltase, contributes to its relevant role. These enzymes facilitate starch degradation and sugar conversion during fermentation, positively impacting kefir’s sensory characteristics and quality [59,60].

Alpha and Beta Diversity

Figure 3a,b shows the alpha diversity analysis for the bacterial and fungal genera, respectively. There is an increase in bacterial diversity, although without significant changes in the dominance of species. This increase may be associated with factors such as specific fermentation conditions, ingredients used, and production practices, suggesting potential health benefits and improvements in the sensory characteristics of kefir [8].
In contrast, the diversity and dominance of fungal species decreases over time, as illustrated in Figure 3b. This reduction can be explained by factors such as the competition between fungi, storage conditions, and degradation of the microbial community, highlighting the importance of maintaining the stability of the fungal microbiota to guarantee the final quality of the product [61].
In the beta diversity analysis, it can be seen that the groups are distant from each other, showing that the maturation time actively affects the bacterial and fungal communities of kefir. The results indicate an instability in the communities throughout the shelf life, making them more susceptible to stress events [62].

3.2. Analysis of Kefir Volatiles

In the group of alcohols, compounds such as 2,3-butanediol and 2-heptanol were detected consistently in all analysis periods (0, 15, and 30 days) (Figure 4). 2,3-butanediol, which has a fatty aroma [63] and is produced by LAB through the butanediol fermentation pathway, is recognized as one of the key aroma components of fermented milks, along with ethanal [64,65].
In turn, 2-heptanol contributes earthy, oily, and mushroomy aromatic notes [63,66], Similarly, 2-heptanol may be related to lipolysis, offering a green and floral aroma to the product [67]. It also contributes a fresh, lemon-like aroma, as well as a herbaceous and grassy scent, accompanied by a subtle ethereal and oily note [68]. Phenylethyl alcohol, detected only after 30 days of fermentation, is known for its floral aroma, reminiscent of rose, violet, and honey, with a spicy touch [69], as well as its antimicrobial potential against filamentous fungi and Gram-negative bacteria [70].
The formation of phenylethyl alcohol is associated with the metabolism of yeasts, particularly species such as Saccharomyces cerevisiae, Kluyveromyces marxianus, Wickerhamomyces anomalus, Pichia kudriavzevii, Pichia farinosa, and Pichia anomala [70,71]. These volatile alcohols represent relevant aromatic compounds in milk kefir, derived from glucose hydrolysis and amino acid metabolism mediated by bacteria and fungi during fermentation [72]. Although ethanol is a common compound in kefir, its concentration remained low in this study, at less than 0.1%. However, it is well-documented that the presence of high levels of higher alcohols can impair the aroma of fermented beverages [73].
With regard to ketones, 2-heptanone, with a characteristic blue cheese aroma and spicy notes, was detected at all stages of the study. Its presence is attributed to lipid metabolism, particularly the lipolytic activity of the lactic acid microbiota [69,74]. 2-nonanone, associated with a ripened, fruity, and smoky cheese aroma, was observed only at the beginning (0 days) [69,75]. On the other hand, 2-undecanone appeared from the 15th day of fermentation.
These ketones result from the metabolism of lipids and carbohydrates, such as lactose, by the bacteria and fungi present [76]. Because they have low perception thresholds, ketones like acetoin contribute intensely to the aromatic profile, giving the drink positive sensory characteristics often associated with a cheese flavor [48,76,77]. The presence of these ketones, in addition to improving the aroma, benefits the overall quality of kefir [78].
With regard to carboxylic acids, butanoic acid and its methylated derivatives, such as 2-methylbutanoic acid and 3-methylbutanoic acid, have been identified only in the early stages of kefir fermentation [63]. These acids, which are products of carbohydrate and lipid metabolism, stood out as the most abundant volatile compounds in kefir samples, although their concentration varied significantly between samples [79].
The carboxylic acids detected later, such as hexanoic acid (characterized by olfactory notes of ripened, rancid, and buttery cheese), n-decanoic acid (with nuances of wax, fruit, and cheese), and octanoic acid (with a sensory profile that includes notes of cheese, sweat, and vegetable), were detected from the 15th day of fermentation and remained until the end of the analysis period [69,80]. On the other hand, the 2-hydroxyethyl propanoic acid ester was only detected at the end of the fermentation period (30 days), indicating late formation. These organic acids, resulting from lipid and carbohydrate metabolism, play an essential role in the sensory characteristics of kefir [79].
Esters, formed by esterification reactions involving alcohols, contribute significantly to the aromatic profile of fermented dairy products [81]. It was observed that the diol 4,5-octanediol, 2,7-dimethyl was exclusive to the beginning of the fermentation process, demonstrating a specific temporal dynamic between volatile compounds.
Over the lifetime of the kefir, a general downward trend in the abundance of the volatile compounds was observed, as measured by the area percentage. An exception to this trend was 2-heptanol, which increased in concentration between days 0 and 15, followed by a reduction at 30 days. This temporal variation suggests a dynamic pattern in the release of volatiles, which may influence the sensory characteristics of kefir throughout storage.

3.3. Correlation Between the Abundance of Microorganisms and Volatiles

The bacterial genera Enterococcus and Acetobacter show positive correlations with various volatile compounds, indicating an important contribution to the complex aroma of kefir (Figure 5).
In particular, the correlation of Enterococcus with 2-heptanol and 2,3-butanediol suggests its direct role in defining specific aromas [82]. In addition, the yeast genus Kazachstania is associated with compounds with fruity and steric notes, enriching the flavor of kefir. The genera Lactobacillus and Lactococcus, known for their central role in fermentation, correlate with compounds such as phenylethyl alcohol, which imparts aromas such as impure, pink, violet, honey, floral, and spicy [69]. Similarly, Walsh et al. (2016) [69] identified a correlation between Lactobacillus kefiranofaciens and the compound phenylethyl alcohol in kefir.
In contrast, negative correlations have been identified between Aspergillus and 4,5-Octanediol, 2,7-dimethyl-; phenylethyl alcohol; octanoic acid; 2-Undecanone; and hezanoic acid, as well as between Meyerozyma and n-decanoic acid; butanoic acid (pungent, cheesy, sour, and rancid taste), 3-methyl-; butanoic acid, 2-methyl-; 2-Heptanone (musty ripened cheese odor) [75]; 2-Nonanone (malt, fruity, hot milk, and smoked cheese); and 4,5-Octanediol, 2,7-dimethyl. These findings suggest that an excessive abundance of these microorganisms can impair the sensory quality of kefir, highlighting the significant impact of yeasts on its volatile compounds.
Unidentified microorganisms (Unknown_Bacteria and Unknown_Fungi) also show moderate correlations with volatile compounds, indicating a possible role in aroma formation, although their specific function is still unclear. Previous studies confirm these correlations, as observed by Dertli and Çon (2017) [79] and Walsh et al. (2016) [69], who associate specific microbial species with the production of volatile compounds and identify genes potentially linked to kefir’s intestinal health benefits.
These correlations between microorganisms and volatile compounds are fundamental for optimizing kefir’s physicochemical and sensory properties. The selection of microorganisms that promote a desirable flavor and aroma allows the control of microbial succession, improving the product quality and allowing the development of differentiated kefirs with a longer shelf life [80].

3.4. Physical and Chemical Analysis of Kefir Throughout Its Shelf Life

The artisanal kefir produced in this experiment was subjected to a physicochemical analysis throughout its storage period, with evaluations carried out on days 0, 15, and 30. The data obtained from these analyses is shown in Table 1.
The pH of the kefir remained stable throughout its shelf life, with a slight variation from 4.40 at the start of maturation to 4.25 at the end of the 30 days. The titratable acidity remained within the maximum limit set by Brazilian legislation (1.0 g/100 g) [15] throughout storage. The acidification of the product results from the conversion of lactose into lactic acid by lactic acid bacteria during fermentation, reducing the pH and increasing the acidity [4,84].
In comparison, Gul et al. (2015) [85] observed a titratable acidity between 0.64 and 0.76 g of lactic acid in kefir after 24 h of fermentation, while Windayani et al. (2019) [86] reported higher values, between 1.96 and 2.64 g of lactic acid, with the same fermentation period. In artisanal kefir, Bilac et al. (2021) [87] found an acidity of 1.03 to 1.10 g of lactic acid after 24 h, increasing to 1.70 to 1.71 g after 48 h, accompanied by a drop in pH from 6.60 to between 4.58 and 4.38 after 24 h, and to a range of 3.74 to 3.60 after 48 h of fermentation.
The concentration of lactose in matured kefir remained at 4.01 g/100g, increasing slightly to 4.03 g/100g after 30 days. Lactose acts as an essential substrate for the kefir microbiota and is converted into lactic acid and other metabolites during fermentation, contributing to the sensory and nutritional properties of the product [7]. β-galactosidase hydrolyzes lactose into glucose and galactose, which are subsequently transformed into lactic acid, potentially making kefir more tolerable for individuals with lactose intolerance [1]. The amount of residual lactose is affected by the composition of the milk and any fortifications [9]; products with a lower lactose content and high microbial counts are more suitable for intolerant consumers [88]. Sainz et al. (2020) [88] observed a greater reduction in lactose in samples with a significant growth of lactic acid bacteria and yeasts.
The absence of ethanol in the samples contrasts with previous studies: Sainz et al. (2020) [89] detected 0.036% ethanol in artisanal kefir with frozen grains from Costa Rica, while Windayani et al. (2019) [86] reported concentrations between 0.38% and 0.59%. The alcohol content in kefir ranges from 0.0 to 0.1 g/100 mL with commercial cultures and from 0.03 to 1.8 g/100 mL with artisanal grains.
The fat content in kefir was 2.54 g/100 g after maturation, increasing to 2.60 g/100 g at the end of 30 days, representing a reduction compared to the UAT whole milk used (3.0 g/100 g) [15]. Dias et al. (2020) [90] pointed out that lipid variations in kefir can be attributed to the fat content of the milk used. Alves et al. (2018) [89] found contents ranging from 1.63% to 2.90%, influenced by the initial fat concentration of the milk. The reduction in lipids (0.46 g/100 g) is related to the action of lipases in the kefir grains and the microbial activity that uses lipids as a source of energy [88,91].
After maturation, the ash content in kefir was 0.831%, decreasing to 0.827% after 30 days, while the protein content increased from 5.27% to 5.25%, compared to whole milk (3.1 g/100g of protein). Vieira et al. (2015) [92] reported that kefir can reach higher protein levels than fresh milk (up to 5.95%). The coagulation of milk proteins, such as casein, by the action of lactic acid bacteria contributes to the high protein content of kefir. Table 2 shows the analysis of kefir’s color and total solids throughout its shelf life.
The luminosity (L) of the UAT milk and kefir grains showed initial values of 74.11 ± 0.64 and 77.19 ± 0.22, respectively, and increased progressively throughout the kefir’s shelf life, reaching 85.52 ± 0.52 after 30 days. The red–green color coordinate (a*) indicated a predominantly greenish trend, varying from −3.42 ± 0.15 on day 0 to −1.09 ± 2.94 on the 30th day of storage. These data contrast with those of Garofalo et al. (2020) [93], who recorded lower average values (−7.90 and −8.48) in artisanal kefir from Bosnia and Herzegovina.
The yellow–blue coordinate (b*) remained in the yellow range throughout storage, reaching 8.69 ± 0.27 after 30 days. As for total solids, the initial milk had 12.24%, while the kefir ranged from 10.09 ± 0.19 on day 0 to 8.69 ± 0.27 at the end of storage. These values are consistent with those found by Gul et al. (2018) [85] in cow’s milk kefir, both in commercial and artisanal versions.

4. Conclusions

This study provided a comprehensive analysis of the microbiota evolution and physicochemical parameters of artisanal kefir during storage, offering valuable insights into the microbial dynamics and their implications for product quality. The findings revealed that Enterococcus and Acetobacter decrease in abundance over time, while Lactobacillus, Lactococcus, Lentilactobacillus, and Leuconostoc remain stable. Kazachstania exhibited the highest initial prevalence among fungi, whereas Penicillium and Aspergillus gradually increased. The bacterial diversity expanded without significant shifts in dominance, suggesting potential health and flavor benefits, while the fungal diversity decreased, highlighting the importance of microbial stability.
Volatile compounds generally declined over time, except for an increase in 2-heptanol, which enriched the aromatic profile with alcohols and ketones. Correlations indicated that Enterococcus plays a key role in kefir aroma, while Aspergillus and Meyerozyma were negatively associated with its sensory quality. The physicochemical analyses confirmed that the pH remained stable, and the titratable acidity was within the legal limits throughout storage.
This study highlights the properties of artisanal kefir in Brazil, emphasizing its compliance with Brazilian legislation and its potential for standardization and commercialization. The maintenance of physicochemical parameters within regulatory limits confirms that artisanal kefir can be safely marketed for up to 30 days. Variations in volatile compounds, particularly the increase in 2-heptanol, highlight the need for further research in their impact on sensory quality and consumer acceptance.
Future studies should explore the relationship between the microbiota, physicochemical attributes, and sensory perception, incorporating detailed sensory analyses and strategies to enhance kefir stability. Consumer education and engagement initiatives can promote kefir consumption and support its market expansion. While industrializing artisanal kefir presents challenges, this study demonstrates that, with Good Manufacturing Practices (GMPs) and process adjustments, it is possible to ensure microbiological stability and maintain product quality over time.

Author Contributions

Conceptualization, J.A.S.; Validation, W.d.C.O., S.H.F. and P.d.S.M.; Formal analysis, A.S.d.F.; Investigation, J.A.S. and A.S.d.F.; Writing—original draft, J.A.S. and M.M.S.; Writing—review & editing, J.A.S., W.d.C.O., A.S.d.F., M.M.S. and S.H.F.; Visualization, M.M.S.; Project administration, P.d.S.M.; Funding acquisition, P.d.S.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq (grant registration number 140557/2024-5).

Data Availability Statement

The sequencing data generated for this study have been deposited in the Sequence Read Archive (SRA) under accession number PRJNA1213721.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 11 00105 g001
Figure 1. Production flowchart for artisanal kefir and analysis of its quality attributes throughout its shelf life.
Figure 1. Production flowchart for artisanal kefir and analysis of its quality attributes throughout its shelf life.
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Fermentation 11 00105 g002
Figure 2. Relative bacterial (a) and fungal (b) abundance of kefir throughout its shelf life.
Figure 2. Relative bacterial (a) and fungal (b) abundance of kefir throughout its shelf life.
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Figure 3. Bacterial (a) and fungal (b) alpha diversity, and bacterial (c) and fungal (d) beta diversity of kefir throughout its shelf life.
Figure 3. Bacterial (a) and fungal (b) alpha diversity, and bacterial (c) and fungal (d) beta diversity of kefir throughout its shelf life.
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Figure 4. Abundance of the volatile compounds identified during kefir’s shelf life (0, 15, and 30 days) in relation to their percentage area.
Figure 4. Abundance of the volatile compounds identified during kefir’s shelf life (0, 15, and 30 days) in relation to their percentage area.
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Figure 5. Hierarchically clustered heatmap of correlations between mold-related strains, bacteria, and fungi, and the levels of volatile compounds in kefir over its shelf life.
Figure 5. Hierarchically clustered heatmap of correlations between mold-related strains, bacteria, and fungi, and the levels of volatile compounds in kefir over its shelf life.
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Table 1. Physico-chemical attributes of artisanal kefir throughout its shelf life.
Table 1. Physico-chemical attributes of artisanal kefir throughout its shelf life.
Parameter0 Days15 Days30 DaysLegal Parameter **
pH4.40 a ± 0.014.32 a ± 0.014.25 a ± 0.02-
Titratable acidity (g lactic acid)0.87 a ± 0.020.92 a ± 0.050.95 a ± 0.05<1.0
Lactose (g/100 g)4.01 a ± 0.014.05 a ± 0.014.03 a ± 0.01-
Ethanol (%)<0.1 a ± 0.00<0.1 a ± 0.00<0.1 a ± 0.000.5 to 1.5
Fat (g/100 g)2.65 a ± 0.052.65 a ± 0.002.60 a ± 0.000.5 to 5.9
Ash (%)0.831 a ± 0.020.834 a ± 0.010.827 a ± 0.01-
Proteins (g/100 g)5.27 a ± 0.055.22 a ± 0.115.25 a ± 0.08Min. 2.9
Humidity90.11a ± 0.3889.30 a ± 0.4789.22 a ± 0.63-
Source: Adapted from Brazil (2007) [15] and FAO/WHO (2003) [83]. Equal letters on the same line indicate that there is no statistical difference. ** Mean values and standard deviations were obtained from triplicate analyses.
Table 2. Analysis of the color and total solids of milk, kefir grains, and kefir throughout its shelf life.
Table 2. Analysis of the color and total solids of milk, kefir grains, and kefir throughout its shelf life.
ColorTotal Solids
VariableL*a*b*
Milk74.11 a ± 0.64−2.41 a ± 0.0520.93 ± 0.3312.24 a ± 0.11
Grain77.19 a ± 0.22−0.85 a ± 2.2821.76 a ± 0.07-
0 days88.03 a ± 0.84−3.42 a ± 0.1510.09 a ± 0.195.95 b ± 0.23
15 days86.45 a ± 0.86−3.40 a ± 0.179.57 a ± 0.605.65 b ± 0.18
30 days85.52 a ± 0.90−1.09 a ± 2.948.69 a ± 0.276.15 b ± 0.15
Equal letters on the same line indicate that there is no statistical difference. Mean values and standard deviations were obtained from triplicate analyses. L* represents luminosity, while a* and b* correspond to chromaticity coordinates.
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MDPI and ACS Style

Ströher, J.A.; Oliveira, W.d.C.; Freitas, A.S.d.; Salazar, M.M.; Flôres, S.H.; Malheiros, P.d.S. Microbial Dynamics and Volatile Compound Profiles in Artisanal Kefir During Storage. Fermentation 2025, 11, 105. https://doi.org/10.3390/fermentation11020105

AMA Style

Ströher JA, Oliveira WdC, Freitas ASd, Salazar MM, Flôres SH, Malheiros PdS. Microbial Dynamics and Volatile Compound Profiles in Artisanal Kefir During Storage. Fermentation. 2025; 11(2):105. https://doi.org/10.3390/fermentation11020105

Chicago/Turabian Style

Ströher, Jeferson Aloísio, Wemerson de Castro Oliveira, Anderson Santos de Freitas, Marcela Mendes Salazar, Simone Hickmann Flôres, and Patrícia da Silva Malheiros. 2025. "Microbial Dynamics and Volatile Compound Profiles in Artisanal Kefir During Storage" Fermentation 11, no. 2: 105. https://doi.org/10.3390/fermentation11020105

APA Style

Ströher, J. A., Oliveira, W. d. C., Freitas, A. S. d., Salazar, M. M., Flôres, S. H., & Malheiros, P. d. S. (2025). Microbial Dynamics and Volatile Compound Profiles in Artisanal Kefir During Storage. Fermentation, 11(2), 105. https://doi.org/10.3390/fermentation11020105

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