The Effects of Non-Viable Probiotic Lactobacillus paracasei on the Biotechnological Properties of Saccharomyces cerevisiae

Abstract

Due to the increasing interest in probiotic components to improve quality of life, this study aimed to investigate the bioactive potential of a paraprobiotic derived from a selected strain of probiotic lactic acid bacteria (Lacticaseibacillus paracasei MIUG BL80) on Saccharomyces cerevisiae MIUG D129, used as a cellular model organism. The paraprobiotics (inactivated cells) were obtained through a combination of ultrasonic and conventional heat treatments. It was observed that adding more than 10 % of the paraprobiotic suspension to the cultivation medium of yeast had a positive influence on the metabolic activity of the starter culture (S. cerevisiae). The specific growth rate increased from 0.227 in the control sample to 0.507 in the sample with 15% paraprobiotic supplementation (S3), while the generation time decreased from 4.403 h to 1.972 h. This suggests that adding probiotics to the cultivation medium enhances the metabolic performance of S. cerevisiae cells. Additionally, an improvement in yeast cell viability during wet biomass storage (from 48 h to 14 days at 4 °C) was observed.

1. Introduction

Natural fermented products contain complex and beneficial microbial communities, including yeasts and bacteria (lactic acid bacteria, acetic bacteria). Yeast–bacteria interactions can be either stimulatory or inhibitory, depending on the species and strain. Yeasts often enhance the stability of lactic acid bacteria (LAB), though the effect depends on both yeast and LAB species. Recent studies, such as one using Saccharomyces cerevisiae in milk kefir, show that yeast can influence ethanol production and sugar utilization [1]. However, not all LAB strains respond the same way, suggesting strain-specific mechanisms. Most tested yeasts improved LAB stability, with comparable effects observed across different S. cerevisiae strains. By contrast, LAB showed more variable responses to yeast co-cultivation [2]. Furthermore, LAB influence yeast cell growth primarily through competition for vital space—a key aspect of biological rivalry—especially since Lactobacillus species proliferate rapidly in the already dense environment of S. cerevisiae. LAB also compete with S. cerevisiae for essential micronutrients required for growth and product synthesis. Importantly, LAB produce end-products such as lactic and acetic acids that inhibit yeast growth. These acids lower cytoplasmic pH, disrupt yeast intracellular metabolism, and induce stress during fermentation [3].

Yeasts and LAB are commonly found in many fermented foods and in the human gut microbiota. In particular, S. cerevisiae and Lactobacillus spp. strains—widely recognized for their health benefits and as key components of traditional fermented foods—often coexist and thrive together [4]. LAB produce metabolites such as carbon dioxide, pyruvate, propionate, and succinate that can support yeast growth. Meanwhile, yeast synthesize vitamins and amino acids that promote LAB development. However, some studies suggest that yeast and LAB strains can also inhibit one another’s growth. For example, LAB-derived compounds, such as 4-hydroxyphenyl lactic acid and cyclic peptides, can suppress yeast-produced substances such as alcohol and fatty acids, which in turn may inhibit LAB [5].

Yeasts are commonly used in the preparation of foods and beverages. Besides their technological and functional properties, they also provide various health benefits and promote well-being. Different yeast genera are associated with specific fermented foods. Species of Saccharomyces spp., including S. cerevisiae, S. bayanus, S. kluyveri, S. rosinii, and S. boulardii, have been isolated from a range of fermented dairy products such as yogurt, cheese, kefir, fermented fish, and beverages. These strains demonstrate survival in gastric juice and bile acid, along with good hydrophobicity, antimicrobial activities, auto-aggregation, and adhesion [6]. Additionally, Saccharomyces spp., particularly S. cerevisiae and S. boulardii, play a beneficial role in the gut microbiota by increasing phylum Bacteroidetes while decreasing the proportions of Firmicutes and Proteobacteria. Yeasts help control inflammation by promoting immune response and boosting short-chain fatty acid production. S. cerevisiae is a prevalent component of the intestinal microbiota, present in over 96% of individuals, and, together with fungi such as Malassezia spp. and Candida spp., contributes to host homeostasis. Moreover, Saccharomyces spp. produce bioactive metabolites that may have medicinal and therapeutic potential [7].

Some yeast species also possess probiotic potential and play a vital role in regulating gut immunity by competing with harmful bacteria and interacting with immune cells. Saccharomyces spp., particularly S. boulardii and S. cerevisiae, are recognized for their antimicrobial, immune-modulating, and gut-restorative properties. Additionally, other yeast genera, such as Debaryomyces spp., Pichia spp., and Yarrowia spp., are gaining attention. Globally, interest in probiotic yeasts has increased, particularly in fermented foods that enhance flavor and nutritional value. Yeasts can boost B-vitamin levels, facilitate mineral absorption, and promote digestion by improving nutrient uptake [8].

Among the many studies on functional fermented foods and their microbiota, the roles of metabiotics (probiotics, prebiotics, postbiotics, and paraprobiotics) have been explored [9]. Probiotics are live microorganisms that, when properly prepared and administered, confer health benefits to the host. Prebiotics are non-digestible food components that are selectively utilized by host microorganisms to produce positive effects. The combination of probiotics and prebiotics is known as synbiotics, which aim to promote the growth or activity of specific health-enhancing bacteria. Paraprobiotics are non-viable, intact, or fragmented microbial cells that, despite losing viability, still provide health benefits. By contrast, postbiotics are soluble bioactive compounds produced or released by bacteria that also confer health benefits [10]. Both paraprobiotics and postbiotics do not contain live cells; instead, they consist of non-viable cells, cell fragments, microbial metabolites (such as organic acids, bioactive peptides, exopolysaccharides, or enzymes), which can still benefit host health. Importantly, they are easier to apply in food systems since they do not require live cells [11,12]. Lactobacillus species primarily used to produce paraprobiotics include Lactobacillus rhamnosus, Lactiplantibacillus plantarum, Limosilactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus acidophilus [13,14,15].

Postbiotics and non-viable cells derived from the probiotic LAB can protect yeast cells during fermentation while enhancing their metabolic activity, owing to their antioxidant, anti-inflammatory, and immunomodulatory effects [16]. Thus, they promote the growth of S. cerevisiae baker’s yeast and increase carbon dioxide production. Furthermore, they support continuous fermentation by boosting yeast resistance to environmental stress and contamination. In baked products, these interactions help optimize fermentation and improve final product quality [17,18].

Saccharomyces spp. are becoming increasingly important in both pharmaceutical and industrial sectors. These yeasts are commonly used to study the stress responses of eukaryotic cells [19]. Yeasts offers several benefits for large-scale production, such as low-cost culture media and a proven track record of efficient fermentation. Additionally, as a by-product of production, yeast biomass serves as a valuable resource for animal feed, without adding disposal costs [20]. Similarly, the Lactobacillus spp. is well known for its biotic (pre-, pr-, post- and parabiotic) benefits. Furthermore, Lc. paracasei species has been granted a qualified presumption of safety by the European Food Safety Authority. Research shows that heat-killed Lc. paracasei has been safely used in human foods for many years and has proven safe in both single-dose and long-term repeated safety tests. Heat-killed Lc. paracasei has demonstrated beneficial effects on human health, including immune-boosting properties, a favorable safety profile, and applications in commercial products such as yogurt, fermented foods, and dietary supplements [21].

The yeast S. cerevisiae is widely used in studies due to its ease of isolation from various sources, its simplicity, and its similarities to eukaryotic cells [22]. Many studies focus on the effect of live Lactobacillus cells on yeasts in the fermentation process, often noting that they can inhibit one another’s growth [23,24]. However, nonviable cells of this bacterium can also help prevent interactions that inhibit yeast growth. Thus, paraprobiotics may be explored to evaluate their effects on yeast cell viability, oxidative stress response, gene expression, and membrane integrity. Components of paraprobiotics, such as peptides or polysaccharides from the cell wall, can interact with yeast cells to activate defense mechanisms or neutralize free radicals. This approach offers a rapid and effective means of assessing the safety and benefits of paraprobiotics, thereby supporting the development of functional products in food, pharmaceuticals, and biotechnology.

This study aimed to evaluate the effect of Lc. paracasei paraprobiotics (produced through combined ultrasonic and conventional heat treatments) on the metabolism of S. cerevisiae by assessing growth kinetics, fermentation capacity, and preservation of viability during wet biomass storage. The results of inactivated cell probiotic bacteria on yeast can be further applied to fermented food and feed formulations, with potential to improve biotication effects.

2. Materials and Methods

2.1. Evaluation of the Functional Efficacy of Paraprobiotics Using the Yeast Model

• Obtaining Paraprobiotics

The probiotic LAB strain Lc. paracasei MIUG BL80 from the MIUG Microorganisms Collection (www.mirri.org), previously isolated from water kefir grain microbiota and preserved in 40% (w/v) glycerol (Scharlau, Sentmenat, Spain) at −80 °C, was reactivated by transferring the contents from a 2 mL Eppendorf tube into 9 mL of sterile MRS broth (Millipore, Buchs, Switzerland) and incubating for 48 h at 37 °C. Then, 10 μL of this culture was streaked onto MRS agar (Millipore, Buchs, Switzerland) to obtain single colonies. Biomass from a single colony was transferred to 50 mL of liquid MRS medium and cultivated in a stationary system for 48 h at 37 °C [23]. The culture was centrifuged at 7000 rpm for 10 min at 4 °C, then washed three times with phosphate-buffered saline (PBS) containing 8 g/L sodium chloride, 0.2 g/L potassium chloride, 1.44 g/L disodium phosphate, and 0.24 g/L monopotassium phosphate, the mentioned reagent supplied by Chimreactiv (Bucharest, Romania), pH 7.2 [24]. The final cell suspension was adjusted to an optical density of 2.0 at 600 nm. This suspension was transferred into a sterile Falcon tube (kept on ice during processing) and sonicated with an ultrasonic generator UP 100H (Hielscher Ultrasonics GmBH, Teltow, Germany), set at 100 W, 30 kHz, pulsation 1, for 20 min at 80% amplitude. After sonication, the suspensions were kept on ice in sterile Falcon tubes until heat treatment at 75 °C for 30 min using the F33MC circulator (water bath) from Julabo Labortechnik GmbH (Seelbach, Germany) [25]. To confirm complete inactivation of cells, 1 mL of the cell suspension was homogenized with 9 mL of sterile saline solution (0.9% NaCl) for decimal dilutions. From the two most adequate dilutions, 1 mL of suspension was aseptically transferred into 20 mL of MRS agar supplemented with 20 g/L CaCO₃ (Chimreactiv, Bucharest, Romania) [26]. The samples were incubated at 37 °C for 72 h, and the absence of colonies was verified [27].

• Obtaining the Yeast Cell Suspension

The yeast strain S. cerevisiae MIUG D129 from the MIUG Microorganisms Collection (www.mirri.org) was stored on solid YGC medium (Yeast Glucose Chloramphenicol Agar) at 4 °C and revived by cultivation on fresh YGC medium at 25 °C for 72 h [28]. Using a sterile loop, the reactivated culture was transferred to 50 mL of sterile 0.9% sodium chloride solution. Yeast cells were then counted with a Thoma cytometer (Marienfeld, Germany) and adjusted to the desired concentration of 1 × 10⁸ CFU/mL suspension.

• Conditions for Yeast Multiplication

Yeast multiplication was carried out by culturing cells in liquid medium as follows: four Erlenmeyer vessels, each containing 200 mL of YPD medium (10 g yeast extract (Sigma-Aldrich, Saint Louis, MO, USA), 20 g/L peptone, 20 g/L dextrose Merck Millipore (Darmstadt, Germany), 20 g/L agar (Oxoid Ltd., Hampshire, UK), were inoculated with 8 mL of yeast inoculum (4%), resulting in 200 mL of culture with an initial concentration of 1 × 10⁶ CFU/mL medium. Three of the four vessels were supplemented with different volumes of paraprobiotics: 5% (S1), 10% (S2), and 15% (S3). The control sample (M) contained no paraprobiotics. The inoculated samples were incubated in a rotary shaker at 200 rpm and at a temperature of 28 °C ± 1 °C for 48 h [29].

• Determination of Dry Matter Biomass Yield

The culture was centrifuged at 6000 rpm for 10 min at 4 °C. The supernatant was discarded, and the culture was washed three times with phosphate-buffered saline (PBS) and centrifuged again under the same conditions. The wet biomass obtained from 200 mL of liquid sample was weighed. A representative portion of the wet biomass was dried at 105 °C in an AND MF-50 moisture analyzer (A&D Company Limited, Tokyo, Japan) until constant weight. The amount of dry matter biomass (g d.m./L) and the yield, expressed as grams of dry matter biomass per liter of culture (g d.m./L), were then determined [30].

• Parameters of Yeast Multiplication in the Presence of Paraprobiotics

The effect of paraprobiotic on yeast growth was monitored by direct microscopic cytometry with a Thoma cytometer (Marienfeld, Germany) under a microscope (OBS, Kern, Germany). Colony-forming units per mL (CFU/mL) were calculated, and a microbial population growth as a function of time was modeled using the reparameterized Gompertz equation, proposed by Zwietering [30,31]:

y = A × exp {−exp [(µ_max × e)/A × (λ + t) + 1

(1)

where y = ln (N/N₀), N₀ is cell concentration after inoculation, CFU/mL; N is cell concentration at the growth time t (t); CFU/mL; A = ln (N$\infty$/N₀) is the maximum value reached with N∞ as the maximum asymptotic population; μ_max is the maximum specific multiplication rate; e is Euler’s number; λ is period of the latency phase.

For the Gompertz modeling, time was measured in hours (6, 12, 24, and 48 h).

After 6 h, 12 h, 24 h, and 48 h of cultivation, the number of viable cells and kinetic parameters of multiplication were determined: multiplication rate (v), generation time (tg), number of generations (n), and yield of dry matter biomass.

Equations (2)–(4) were used for kinetic parameters:

n = (log N—log N₀)/log2

(2)

where N is cell concentration after cultivation time (t), CFU/mL; N₀ is cell concentration after inoculation, CFU/mL;

v = n/t

(3)

and t is cultivation time (h).

t_g = 1/v

(4)

where v is multiplication rate (h⁻¹).

• Evaluation of Yeast Cell Viability

A cell viability test was performed by microscopic examination in the presence of the methylene blue indicator, based on the ability of viable cells to reduce the redox indicator from its oxidized blue form to the colorless reduced form of the leukoderivative [30].

The percentage of viable cells was calculated as

Cell viability (%) = N₀/N_t × 100%

(5)

where N₀ is number of live cells that appear clear after the stain; N_t is the total number of observed yeast cells.

• Evaluation of Yeast Stability During Storage

To assess the influence of paraprobiotic supplementation on yeast survival during refrigerated storage, samples with paraprobiotic coded S1, S2, S3, and M were stored in a refrigerator at 4 °C. Cell viability was measured after 48 h, 7 days, and 14 days of storage using Equation (5).

• Evaluation of the Fermentative Capacity of Yeast

The effect of paraprobiotic supplementation on the fermentative capacity of the yeast S. cerevisiae was determined using the Ostrovski method, with some modifications [32,33,34]. Dough balls were prepared from 7.5 g of flour and 5 mL of inoculum suspension from the samples obtained above, coded M, S1, S2, and S3, then kneaded for 5 min. Each dough ball was immersed in a Berzelius beaker containing 200 mL of water at a temperature of 32 °C, and the time required for the ball to rise to the surface was recorded. During fermentation, yeast metabolizes carbohydrates in the dough, releasing carbon dioxide (CO₂), which forms bubbles that cause the dough balls to become lighter and cause them to rise to the surface of the water. The time required for the dough balls to float serves as an indicator of the yeast’s fermentative activity: the more active the yeast, the faster the balls reach the surface, reflecting a shorter fermentation time and, implicitly, more intense fermentation activity.

2.2. Statistical Analysis

All data were collected from independent, repeatable experiments, and mean values were calculated from triplicate samples. Statistical significance was assessed using analysis of variance (ANOVA) at a 95% confidence level, with validity confirmed by the regression coefficient R² and the p-value (p < 0.05) through the Tukey test. Minitab 19 (Minitab LLC, State College, PA, USA) was used for statistical analysis.

3. Results

3.1. Evaluation of the Efficacy of Paraprobiotics on the Multiplication of S. cerevisiae

Paraprobiotics produced through combined ultrasound-heat treatment were tested for their effect on S. cerevisiae yeast cells. According to the Gompertz model, the maximum asymptotic population value is shown by parameter A, which increased in samples with added paraprobiotics (from 1.461 for S1 to 1.586 for S3) compared to the control sample (1.334). The growth rate, provided by (µ_max × e)/A parameter, slightly increased with the amount of paraprobiotics, from 195.800 for the M sample to 196.800 for the S3 sample (

Table 1. Yeast cell multiplication parameters according to the Gompertz model.

Parameters	M	S1	S2	S3
A	1.334 ± 0.320	1.461 ± 0.273	1.505 ± 0.243	1.586 ± 0.235
(µmax × e)/A	195.800 ± 5.803	196.300 ± 4.922	196.500 ± 4.374	196.800 ± 4.247
λ	−0.010
R2	0.538	0.657	0.720	0.752
MSD	0.422

).

In the context of this experiment, the observed R² values (ranging from 0.538 to 0.752) are moderate, indicating that the model explains only partial variation in the fermentation process. Although not ideal, these values are reasonable for a complex system (Table 1).

The Mean Squared Deviation (MSD) parameter is estimated based on the mean error between the observed and predicted values by the model. The MSD value of 0.422 suggests a reasonable fit of the model to the experimental data, considering the complexity of the fermentation process, which involves factors such as temperature, time, pH, substrate composition, gas concentrations (including CO₂, O₂, and N₂), and microbial activity.

The effect of paraprobiotics on the multiplication dynamics of baker’s yeast is illustrated in Figure 1. Experimental data represent the values obtained from the trials, while predicted data are derived from the Gompertz model. At all studied temperatures (65, 75, and 85 °C), the p-values indicate no significant differences between experimental and predicted results (p = 0.995, p = 0.997, and p = 1.000, respectively), as shown by ANOVA and the Tukey post hoc test. The values predicted by the model follow the same general trend as the experimental data. The model helps highlight trends in the experimental results and better estimate the effect of paraprobiotics. Furthermore, in all tests, the exponential phase is accelerated more significantly during the first 6 h. The samples showed similar effects in stimulating cell multiplication, with only slight differences between them; however, in the S3 coded sample (supplemented with 15% paraprobiotic in the cultivation medium), the stimulation was more pronounced compared to other samples.

Table 2. Kinetic parameters of yeast cell multiplication in the exponential phase (6 h).

Sample	Generation Numbers (n)	Multiplication Rate (v), 1/h	Generation Time (tg), h
M	1.363 ± 0.101 b	0.227 ± 0.060 b	4.403 ± 0.474 a
S1	2.252 ± 0.320 ab	0.375 ± 0.101 ab	2.665 ± 0.653 b
S2	2.695 ± 0.379 a	0.449 ± 0.014 a	2.226 ± 0.133 b
S3	3.042 ± 0.536 a	0.507 ± 0.094 a	1.972 ± 0.081 b

Different letters (a, b) in the column indicate significant differences for p < 0.05.

shows the kinetic parameters of yeast growth after 6 h of cultivation during the exponential phase.

Experimental data indicate that paraprobiotics have a positive influence on yeast multiplication kinetics, with the most substantial effect observed at concentrations exceeding 10% (samples S2 and S3). The generation number model has an R² of 0.814 and a p-value of 0.003; the multiplication rate model has an R² of 0.743 and a p-value of 0.010; and the generation time model has an R² of 0.889 and a p-value of 0.000.

After 6 h of cultivation during the exponential growth phase, the number of generations increased from 1.363 in the control sample to 3.042 in the S3 sample, indicating greater cell multiplication in samples with added paraprobiotics. The multiplication rate follows a similar upward trend, increasing from 0.227 in the M sample to 0.507 in the S3 sample. Generation time, the period needed for the cell population to double, decreased significantly from 4.403 h in the M sample to 1.972 h in the S3 sample. This result demonstrates the beneficial effect of supplementing the fermentation medium with 15% LAB paraprobiotics on the multiplication of S. cerevisiae yeast cells.

Additionally, the dry biomass yield after 48 h of cultivation was highest in the control sample (5.078 g/L) and S2 sample (4.955 g/L), followed by the S1 sample (4.780 g/L) and the S3 sample (4.527 g/L). With increasing percentages of paraprobiotic supplementation, the biomass yield decreased. Considering the kinetic parameters of yeast multiplication, it may be assumed that paraprobiotics stimulate yeast cell division and accelerate the cell cycle, resulting in more cells, including newly formed daughter cells, which may be smaller and less metabolically dense, thereby contributing to the reduced dry biomass yield. This effect is particularly useful in applications requiring high cell numbers rather than biomass, such as yeast probiotics, rapid fermentations in baking and beverage production, and basic research on cell cycle regulation and yeast–bacterial interactions. Overall, these findings support the development of faster and more efficient yeast-based biotechnological processes, though further studies are needed to confirm these assumptions.

3.2. Evaluation of the Fermentative Activity of Yeast in the Presence of Paraprobiotics

The Ostrovski method was used to evaluate the fermentative activity of yeast grown in an environment supplemented with paraprobiotics, which involved determining the time required for the dough ball to rise to the surface.

Experimental data also show that increasing the concentration of paraprobiotics enhances the fermentation capacity of the tested yeast. As shown in

Table 3. Evaluation of fermentative activity of yeast by the Ostrovski method.

Sample	M	S1	S2	S3
Pick-up time, min	14.450 ± 0.104 a*	14.200 ± 0.153 a	13.410 ± 0.108 b	13.260 ± 0.168 b

* Different letters (a, b) show significant differences for p < 0.05.

, statistically significant differences were observed between the control sample and the S2 and S3 samples, indicating that supplementation of the fermentation medium with 10%–15% paraprobiotic suspension positively influences the yeast’s fermentation capacity.

3.3. The Effect of Paraprobiotics on Yeast Viability During Wet Biomass Storage

S. cerevisiae is used as a model organism for studying the cellular response to various types of stress [35].

As shown in

Table 4. Viability of yeast cells at 4 °C.

Sample	Viability of Cells, %
	48 h	7 day	14 day
M	90.74 ± 0.63 a*	89.44 ± 0.51 a	88.24 ± 0.44 a
S1	93.11 ± 0.39 b	92.68 ± 1.25 b	91.57 ± 0.83 b
S2	94.48 ± 0.60 c	94.18 ± 0.80 c	94.15 ± 0.64 c
S3	95.57 ± 0.74 d	95.39 ± 0.76 d	95.45 ± 1.25 d

* Lowercase letters (a, b, c, d) in a column denote significant differences between the control sample and the p samples (p ˂ 0.05). The samples consist of yeast combined with varying quantities of paraprobiotics: 5% in S1, 10% in S2, 15% in S3, while M contains no paraprobiotics.

, the control sample maintained a viability of 97.24%, while sample S1 exhibited 98.35% after 14 days. Samples S2 and S3 showed even higher viability, at 99.65% and 99.87%, respectively. These results indicate that the tested paraprobiotic, at concentrations of 10% and 15%, positively influences yeast cell viability during 14-day storage of wet biomass at 4 °C.

4. Discussion

These results provide a basis for interpreting the implications of non-viable LAB probiotic cells in the context of enhancing the yeast biotechnological potential. Further aspects are discussed in detail below. The study found that non-viable cells obtained via the combined ultrasound-heat treatment of Lc. paracasei MIUG BL80, previously isolated from the water kefir grain microbiota, enhanced the growth of S. cerevisiae yeast, with increased maximum population levels observed by cultivation in a specific medium supplemented with paraprobiotics. The exponential growth phase was initially accelerated, especially in samples with over 10% paraprobiotics. While adding 15% paraprobiotic slightly decreased biomass yield, it still had a positive influence on yeast cell division and viability during storage for up to 14 days at 4 °C. Overall, the presence of 10% and 15% paraprobiotics in the fermentation medium promotes yeast metabolic activity and cell viability.

The cell of the yeast S. cerevisiae contains approximately 70–72% water, along with 42–45% protein, 40% carbohydrates, 7.5% lipids (on a dry matter basis), and B vitamins. Analysis of yeast cells reveals that they are a complex biological system that changes over time, with these changes being highly dependent on storage conditions, particularly temperature. For example, a sample stored at 4 °C for 12 days showed a decrease in total carbohydrate content from 48.81% to 37.50% on a dry matter basis, and the trehalose content was measured at 12.33% [36]. Furthermore, another study evaluated the viability of yeast cells stored at temperatures ranging from −20 °C to 30 °C in liquid media supplemented with lactose, starch, and trehalose. After 4 weeks of storage at 4 °C and 10 °C, an unusual strain of Pichia spp. demonstrated a viability of 75–90%, with no significant differences observed in cell stability [37].

Regarding growth kinetics, studies in the literature indicate that S. cerevisiae demonstrates a typical pattern, characterized by a lag phase lasting from 0 to 4 h, followed by an exponential phase from 4 to 12 h, and finally a death phase between 12 and 16 h. The growth rate during this period ranged from 0.344 to 0.367 h⁻¹, with a maximum growth rate of 0.380 h⁻¹ [1]. Additionally, various kinetic models for S. cerevisiae growth during specific fermentation processes have been examined. These models showed a Root Mean Squared Error (RMSE) ranging from 0.1103 to 0.5721, and the coefficient of determination (R²) ranged from 0.81 to 0.97, indicating good fits. The maximum specific growth rate (μ_max) ranged from 0.377 to 0.593 h⁻¹, and S. cerevisiae began visible growth within 3 h after inoculation. The maximum cell dry weight was generally reached at 23 h, after which growth slowed and the cells entered the stationary phase between 24 and 27 h. Subsequently, the cells gradually entered the death phase, although no obvious visual changes were apparent [38].

Moreover, a recent study examined the interaction between S. cerevisiae and LAB during fermentation. Interestingly, the yeast cell yield remained unaffected after 24 h, regardless of the presence of bacteria. However, the maximum specific growth rate (μ_max) was mostly reduced in co-cultures, except when co-cultured with Lactobacillus sanfranciscensis. Specifically, co-cultivation of S. cerevisiae with L. sanfranciscensis resulted in μ_max values of 5.62 for yeast and 5.08 for bacteria, whereas co-culture with L. brevis provided values of 4.03 for yeast and 1.87 for bacteria. Conversely, the combination with L. paralimentarius produced μ_max values of 3.97 and 6.83, respectively. Notably, the final cell yield of LAB was unaffected by the presence of yeast; however, their growth rates varied—L. brevis’s μ_max decreased while L. sanfranciscensis’s increased in co-culture. Minor variations were also observed with L. paralimentarius [39].

Regarding the dynamics of the yeast population, two different strains of S. cerevisiae demonstrated similar behavior during the first 12 h of fermentation. Nevertheless, by 24 h, significant differences became evident: one strain maintained a nearly constant population, whereas the other exhibited a slight increase toward the end. It is well known that LAB can positively influence yeast growth in fermented foods by acidifying the environment; in turn, yeast can provide growth factors such as vitamins and amino acids to LAB. Notably, the viability of Lc. paracasei was higher during storage periods, both in single-culture and co-inoculation assays, over 28 days. Additionally, the yeast strains maintained high viability throughout this storage period [40].

Research also indicates that paraprobiotics are generally more stable than live probiotics, maintaining their key bioactive components even after heat treatment. These studies also show that paraprobiotics are more resilient under stressful conditions than live probiotics. As a result, they serve as effective alternatives, overcoming challenges such as instability during production and long-term storage. Moreover, paraprobiotics offer practical and technological benefits, including an extended shelf life and no requirement for a cold chain, which ensures microbial stability and viability. The existing literature primarily focuses on biotechnological improvements to yeast activity. Importantly, products supplemented with paraprobiotics have scored highest in terms of taste and overall impression, without altering the product’s physicochemical properties. Consequently, manufacturers can produce commercial products containing paraprobiotics without needing to modify the current production process [41].

Regarding the effect of paraprobiotics on eukaryotic cells, paraprobiotics from strains Lc. plantarum, Latilactobacillus sakei, and Limosilactobacillus reuteri, obtained by heat (121 °C, 15 min) and homogenized through sonication for 10 min, showed higher radical scavenging activity (compared to probiotic strains), protected against ROS damage in Caco-2 cells, and enhanced tight junctions. In a colitis mouse model, paraprobiotics—especially from Lc. plantarum and L. reuteri—more effectively improved mucin secretion, reduced inflammation, and restored the gut barrier [42]. However, research on the effects of paraprobiotics derived from lactic acid bacteria on yeast cell metabolism, growth rates, fermentation capacity, and storage stability is limited.

Our preliminary study demonstrates the positive effect of ultrasound-thermal treatments on paraprobiotic derivatives from a probiotic strain (Lc. paracasei MIUG BL 80, isolated from water kefir grains microbiota) on the metabolic functionality and stability of yeast S. cerevisiae. This study presents future opportunities to enhance the behavior of S. cerevisiae as a starter culture and the bioactive content of fermented products. Thus, future directions for paraprobiotic–yeast products involve developing standardized, scalable formulations for food and feed applications. However, it is necessary to clarify paraprobiotic–yeast interactions as well as their mechanisms of action, through investigations of bacterial biochemical components in non-viable cells, molecular mechanisms, strain selection, and evaluation of in vivo benefits.

5. Conclusions

Inactivated bacterial cells or cell fragments of the probiotic strain Lc. Paracasei MIUG BL 80, obtained through combined ultrasonic and conventional heat treatments, positively affected the metabolic behavior (multiplication and alcoholic fermentation) of S. cerevisiae yeast when the cultivation medium was supplemented with paraprobiotic concentrations of 10% and 15%. Moreover, the presence of 15% paraprobiotic during yeast cultivation slowed the autolysis process of yeast cells when wet biomass was stored at 4 °C for 14 days. These findings provide a foundation for practical applications of paraprobiotics to enhance the functionality and stability of yeast starter cultures, as well as to improve the bioactive characteristics of the resulting fermented products. Overall, the acquisition and application of paraprobiotics present new opportunities for advancing bioprocesses and bioproducts aimed at enhancing bioactive properties.