Effect of Commercial Bioprotective Lactic Cultures on Physicochemical, Microbiological, and Textural Properties of Yogurt

Abstract

The present study investigated the effects of the commercial biopreservatives FRESHQ-11 (Lactobacillus rhamnosus), labeled as F, and HOLDBAC YM-B LYO 100 DCU (Lactobacillus rhamnosus and Propionibacterium freudenreichii subsp. shermanii), labeled as H, at different dosages on the pH, titratable acidity (%), fungal inhibition, and textural parameters of yogurt during 28 days of storage at 7 ± 1 °C. The study compared these biopreservatives with yogurt containing only the chemical preservative potassium sorbate at the maximum allowed concentration (C1) and yogurt without any chemical preservatives (C2), with the goal of identifying alternatives to reduce or replace potassium sorbate. Yogurts were formulated with biopreservatives at concentrations of 0.1% and 0.2% (v/v) and with potassium sorbate at 0.015% and 0.03%. The results indicated that yogurts containing biopreservatives had significantly lower pH and higher titratable acidity (%) than C2 (p < 0.05). Syneresis significantly decreased over the 28-day storage period at 7 ± 1 °C (p < 0.05). Additionally, yogurts with bioprotective cultures exhibited significantly lower textural parameters (p < 0.05) compared to C1 and C2. This study underscores the potential of biopreservatives as viable replacements for potassium sorbate, with these formulations being comparable to C1 in inhibiting molds and yeasts, particularly when L. rhamnosus was used at 0.2% v/v. This finding is promising for future pilot and industrial-scale applications.

1. Introduction

Consumers are now more informed about foods and ingredients, especially functional foods, which have sparked considerable interest due to their advantages in preventing various health problems, enhancing physicochemical functions, and high nutritional value [1]. Therefore, several recent studies have shown increased demand for these beneficial natural products [2]. Yogurt is one of the most consumed fermented dairy products due to the combination of pasteurized milk with lactic acid bacteria (LAB), specifically Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus under controlled conditions [2]. Various yogurt formulations are currently being produced to enhance the product’s nutritional value, quality properties, and health properties and meet the growing consumer demand for this type of yogurt [2,3]. Ultimately, adding natural functional ingredients significantly enhances the biological effects of yogurt, including its antioxidant, antidiabetic, anti-inflammatory, antibacterial, and anticancer properties [4].

Preventing deterioration by molds and yeasts in food currently relies on prevention methods (e.g., implementation of Hazard Analysis and Critical Control Points—HACCP), hurdle technologies (e.g., heat treatments, water removal, modified atmosphere packaging, salting, fermentation), and the use of chemical preservatives, including benzoate, propionate, sorbate, nitrate, and sulfites [5]. Agri-food industries primarily depend on chemical preservatives to control microbial contamination and extend product shelf life. However, there is increasing social demand for minimally processed and preservative-free foods, prompting ongoing revisions to additive regulations aimed at limiting their use [6].

Currently, food deterioration caused by the growth of molds and yeasts has become a significant concern worldwide. Between 25 and 40% of food products in various developing countries are lost due to mold and yeast deterioration [7]. Therefore, it is necessary to find alternative strategies to prevent spoilage caused by fungi and/or increase the products’ shelf life [8]. Currently, biopreservation by LAB is the most promising alternative to chemical preservatives in the dairy industry due to their Generally Recognized as Safe (GRAS) status and Qualified Presumption of Safety (QPS) status in the United States and the EU, respectively [9]. Various bioactive metabolites (such as proteinaceous substances, cyclic dipeptides, organic acids, fatty acids, and hydrogen peroxide) generated by LAB are closely associated with the inhibitory effect on molds and yeasts [10]. Salas et al. [11] also report that PAB are considered antifungal. According to Yerlikaya et al. [12], although propionibacteria spp. are used for many purposes, including as a biopreservative culture and adjunct, P. freudenreichii may also be suitable to produce probiotic dairy beverages and does not have adverse effects on the physicochemical, rheological, microbiological, and sensory properties of the product.

However, although commercial antifungal formulations based on LAB and PAB are available for application in different food formats, more detailed research is still needed to understand their effects on final food quality, stability during processing, mechanism of action, synergistic activities, safety, etc., as well as their potential for commercial food exploitation [13]. Additionally, many factors can affect the rheology of yogurt, such as solids content, milk and fermentation thermal treatment temperatures, and homogenization pressure, among others [14]. The types of dairy cultures added during the manufacturing process also influence yogurt rheology.

Numerous studies in the literature have examined the effects of bioprotective cultures on the microbiological, physicochemical, and rheological parameters of fermented milk. However, most of this research has been conducted in developed countries where the raw materials used for fermented milk production are of significantly higher microbiological quality than those typically available in the Brazilian Northeast. In this region, research on bioprotective cultures is scarce or non-existent. Therefore, the conclusions drawn from studies conducted elsewhere require further investigation to assess the efficacy of commercial lactic acid bacteria (LAB) as antifungals under the specific hygienic and sanitary conditions, ambient temperatures, and raw material acquisition processes prevalent in the Brazilian Northeast during yogurt production, processing, and preservation.

Moreover, most commercial bioprotective culture manufacturers currently recommend their use in conjunction with chemical preservatives, primarily potassium sorbate, as an additional food safety measure. These manufacturers do not generally suggest replacing chemical preservatives with bioprotective cultures at the recommended dosages.

The dairy basin in the Brazilian Northeast is mostly composed of small producers who cannot afford to invest in appropriate infrastructure for obtaining milk on their properties, with milking being performed manually under inadequate hygienic conditions. The Brazilian dairy sector has been facing quality issues with its raw materials, repeating errors that demonstrate the need for improvements in handling, cooling, and collection stages, but primarily a cultural change for everyone involved [15]. All these reported problems directly influence the microbiological quality of yogurt made with this raw material, given that, according to Viljoen [16], various factors make the production environment conducive to the development of molds and yeasts in dairy products; among them, the quality of the raw material stands out.

Therefore, in this context of raw material acquisition in the Brazilian Northeast and consumers increasingly seeking natural products without chemical preservatives, this research investigated the effect of the commercial biopreservatives FRESHQ-11 (F) and HOLDBAC YM-B LYO 100 DCU (H) on pH, titratable acidity (%), fungal inhibition, syneresis, and yogurt texture parameters during 28 days of storage at 7 ± 1 °C to obtain new formulations based on their concentrations as an alternative to chemical preservatives.

2. Materials and Methods

2.1. Bioprotective Cultures

A commercial culture composed exclusively of LAB and another culture consisting of a mixture of LAB and dairy PAB with bioprotective properties—Lactobacillus rhamnosus (FreshQ^® 11, Novanesis, Kongens Lyngby, Denmark), provided by Chr. Hansen^®, Valinhos, Brazil, and Lactobacillus rhamnosus and Propionibacterium freudenreichii subsp. shermanii (HOLDBAC YM-B LYO 100 DCU, IFF Health & Biosciences, Dange Saint Romain, France), provided by Fermentech/Danisco Brazil—was utilized. The cultures were prepared for the experiments according to the manufacturer’s recommended dosage, as well as at a doubled dosage. For this purpose, each culture was dissolved in one liter of pasteurized milk at 25 °C, then stored in sterilized 1.5 mL containers and kept frozen at −10 °C for 15 days.

2.2. Experiment Distribution

To study their biopreservative capacity and its effects on yogurt compared to the chemical preservative potassium sorbate, dosages of 0.1% and 0.2% (v/v) were used for both types of commercial bioprotective cultures. Additionally, potassium sorbate was tested at concentrations of 0.015% and 0.03%, as outlined in

Table 1. Final experiment distribution for the study of biopreservative performance.

Exp.	Chemical Preservative Content * (g/100 g)	Commercial Biopreservative Cultures **	Culture Concentration (v/v)
C1	0.030	Absent	0.0%
C2	0	Absent	0.0%
H1	0.015	H	0.1%
F1	0.015	F	0.1%
H2	0	H	0.1%
F2	0	F	0.1%
H3	0	H	0.2%
F3	0	F	0.2%

* Chemical preservative potassium sorbate; ** FRESHQ-11 (L. rhamnosus), referred to as F, and HOLDBAC YM-B LYO 100 DCU (L. rhamnosus and P. freudenreichii subsp. shermanii), referred to as H.

. The C1 and C2 yogurts served as controls for comparison with the yogurts prepared using LAB and dairy PAB cultures.

Similar studies, where the parameters of yogurt containing bioprotective cultures at different dosages were compared with a control yogurt containing only initial cultures, have been conducted by Zhao and Liang [17], Fayyaz et al. [18], and Li et al. [19], among others.

2.3. Yogurt Preparation

In the production of yogurt, whole pasteurized milk sourced from a dairy in the state of Paraíba, located in the Northeast region of Brazil, was used. This milk was analyzed, and the chemical preservative potassium sorbate was added for the C1, H1, and F1 experiments. The milk was then pasteurized again at 86 ± 1 °C for ten minutes, cooled to 42 ± 1 °C, and inoculated with the starter cultures Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus, provided by Chr. Hansen^® Brazil. The biopreservatives were subsequently added in experiments H1, F1, H2, F2, H3, and F3. The mixture was maintained isothermally at 42 ± 1 °C for five to six hours until the pH reached 4.7. The yogurt was then cooled to 20 °C within 15 min, packaged in sterilized 100 mL plastic bottles, and stored at 7 ± 1 °C until physicochemical (pH and titratable acidity) and microbiological (molds and yeasts) analyses were conducted. Finally, the yogurt was packaged in sterilized 300 mL plastic bottles and stored at 7 ± 1 °C.

2.4. pH and Titratable Acidity Analysis

During fermentation, the pH of the yogurts was determined and recorded (using Milwaukee’s MW150 MAX pH meter, Milwaukee Instruments, Rocky Mount, NC, USA) until a pH of 4.70 was reached, and the fermentation time was recorded. The pH was also determined every seven days for a maximum of 28 days. A potentiometric method was used for the determination of the titratable acidity levels expressed in % lactic acid, conducted in triplicate through acid–base titration, using phenolphthalein as the indicator [20].

2.5. Microbiological Analyses (Mold and Yeast Counting)

To conduct mold and yeast analyses in triplicate on Days 1, 7, 14, 21, and 28, CompactDry YM plates (Nissui Pharmaceutical Co., Ltd. Tokyo, Japan), pre-prepared with culture medium, were used to minimize the risk of contamination during the analyses. CompactDry YM plates feature a dry culture medium covered by a layer of chromogenic absorbent tissue, which enhances productivity and efficiency. For each experiment, the samples were diluted to 10⁻¹ and 10⁻² in sterile peptone water, which had been autoclaved at 121 °C for 15 min. Subsequently, 1 mL of each diluted sample was inoculated onto the CompactDry YM plates and incubated at 25 ± 1 °C for seven days. Following the incubation period, colony counts were performed using the Phoenix CP602 colony counter (PHOENIX LUFERCO LTDA, Araraquara, São Paulo, Brazil). According to Brazilian legislation, the maximum acceptable standard for yogurt is a total mold/yeast count of M = 2.0 × 10² CFU/g [21].

2.6. Syneresis Analysis (Whey Separation)

Syneresis analysis of the different yogurt samples stored at 7 ± 1 °C was performed on Days 1, 7, 14, 21, and 28 in quadruplicate by centrifugation according to the methodology proposed by Jauregui et al. [22] with modifications by Beuschel et al. [23]. For this purpose, 5.0 g of the samples was weighed and centrifuged at 2500 rpm for 20 min at 7 ± 1 °C in the refrigerated centrifuge NT 815 (Nova técnica, São Paulo, Brazil). The supernatant was collected and weighed, and the syneresis index was determined according to Equation (1).

$$Syneresis\left(\%\right)={\displaystyle \frac{supernatant weight \left(g\right)}{yogurt total weight \left(g\right)}}\times 100\%$$

(1)

2.7. Apparent Viscosity Measurements

Apparent viscosity measurements were made on yogurt samples using a Brookfield viscometer (RV+ model, Brookfield Engineering Laboratories Inc., Middleborough, MA, USA) at a temperature of 7 ± 1 °C with a spindle speed of 50 rpm. The temperature was maintained using a thermostatically controlled water bath. All data were taken after 30 s for each sample. Appropriate spindles were used, guaranteeing torque within 25 to 75%. A 500 mL beaker was used for all measurements with the guard leg on, and enough sample was added to cover the immersion grooves on the spindle shafts. All experiments were replicated three times. Shear rates were calculated by the method of Mitschka [24,25].

2.8. Texture Parameter Analysis

The instrumental texture of the yogurts was determined using a TA.XT Plus texture analyzer (Stable Micro Systems^®, Godalming, UK). Firmness, consistency, cohesiveness, and viscosity index parameters were evaluated using a compression test employing the A/BE probe with a diameter of 40 mm. Samples were homogenized and placed in an acrylic test cup until reaching a volume of 150 mL (height of 50 mm). The test speed was set to 1 mm/s with a max distance of 30 mm. Sample evaluation was conducted in quadruplicate on Days 1, 7, 14, 21, and 28 at 6.0 to 6.5 °C. As a result of these experiments, force–time curves were built and analyzed to determine the mechanical parameters (Firmness, Consistency, Viscosity Index, Cohesivity) [26].

2.9. Statistical Treatment of Data

Statistical differentiation of the data was performed using one-way analysis of variance (ANOVA). A p-value of ≤0.05 was considered statistically significant for differences among the data groups. Tukey’s test was subsequently applied to evaluate the specific mean differences between groups.

3. Results and Discussion

3.1. Effect of Bioprotective Cultures on pH and Titratable Acidity

The following analysis evaluates the effects of biopreservative cultures and potassium sorbate on the physicochemical, microbiological, and textural properties of yogurt over 28 days of storage. Figure 1 illustrates the variation in pH over the 28-day storage period at 7 ± 1 °C. As shown in the figure, there is a gradual decrease in pH values, which corresponds to the continuous production of organic acids by the starter cultures during storage. This decline in pH is indicative of the ongoing metabolic activity of the lactic acid bacteria, contributing to the yogurt’s acidity over time.

Control yogurt C2 exhibited higher pH values compared to the yogurts containing bioprotective cultures, except for the F2 and H3 experiments, where the pH values were equal to or greater than that of C2 at the conclusion of the storage period. With respect to titratable acidity (%), Figure 2 indicates an inverse relationship with pH, showing a significant increase (p < 0.05) in titratable acidity across all yogurts over the storage period. Among the yogurts containing bioprotective cultures, the H3 sample (L. rhamnosus and P. freudenreichii subsp. shermanii) at a concentration of 0.2% v/v exhibited the lowest titratable acidity throughout the study. This lower acidity is attributed to the ability of propionic acid bacteria (PAB), such as P. freudenreichii, to metabolize lactic acid into various organic acids, including propionic acid and acetic acid [27].

In the C2 yogurt, which contained only the starter Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, significantly lower (p < 0.05) titratable acidity (%) values were observed compared to the yogurts containing bioprotective cultures over the 28-day study period. This pattern, where yogurts with bioprotective cultures exhibited lower pH and significantly higher titratable acidity (%) than C2 yogurt during the 28 days, is consistent with findings by Khan-Mohammadi et al. [28]. In their study, the lowest pH values were observed in T1 (yogurt formulated with starter cultures containing 10⁶ CFU/mL of L. rhamnosus) and T3 (yogurt formulated with starter cultures containing 10⁶ CFU/mL of L. rhamnosus and 10⁵ CFU/mL of Penicillium expansum) throughout the study period. The researchers suggested that this could be attributed to the presence of the probiotic bacterium L. rhamnosus in combination with the yogurt starter cultures. In their study, yogurt T0, which is comparable to C2 in the present research, contained only the starter cultures and similarly exhibited a higher pH than the yogurts with bioprotective cultures.

When comparing yogurt C1 with yogurt C2, it was observed that yogurt C1, which contains the chemical preservative potassium sorbate, exhibited a lower pH and significantly higher titratable acidity (p < 0.05) throughout the 28-day storage period at 7 ± 1 °C. In addition to the cultures used, post-acidification of the yogurt depends on other factors, such as the composition of the raw material, the manufacturing process, and the fermentation time, which was 30 min longer for yogurt C2 than for yogurt C1. Numerous studies have noted that the composition of fermented beverages plays a crucial role in determining the final pH value and its changes during refrigerated storage, which are influenced by the buffering capacity of the beverage, the levels of non-protein nitrogen and vitamins, and the availability of fermentable carbohydrates necessary for the growth of microorganisms [29]. Furthermore, certain species of molds and yeasts can raise the pH of the medium, which may explain the higher pH and lower acidity of yogurt C2, which showed a high level of yeast contamination. Lactic acid bacteria produce organic acids, primarily lactic acid, while yeasts assimilate these acids, forming alcohol and CO₂, thereby increasing the pH. Additionally, during the metabolism of lactic acid, yeasts release vitamins such as vitamin B, pantothenic acid, niacin, riboflavin, and biotin, which are important for the development of lactic acid bacteria [16].

Several studies have demonstrated that LAB, such as L. rhamnosus (F), produce lactic acid and various other organic acids during and after fermentation, which increases the titratable acidity of yogurt and lowers its pH. This creates an environment that is unfavorable for the growth of undesirable microorganisms, including pathogenic bacteria, yeasts, and molds. For instance, Arena et al. [30] found that among the organic acids produced by LAB, lactic acid and acetic acid are the most well-characterized and effective metabolites, particularly in their protonated form at low pH. Similarly, Garnier et al. [31] reported that propionic, lactic, acetic, and butyric acids were the predominant fermentation products in milk fermented with L. rhamnosus and Acidipropionibacterium jensenii. LAB are known for their antimicrobial properties, producing a wide range of antimicrobial substances, including organic acids, fatty acids, reuterin, antifungal peptides, and bacteriocins [32].

In the study by Kariyawasam et al. [33], the titratable acidity of yogurt samples also showed a continuous increase during storage. The titratable acidity values of control C yogurt samples, L. rhamnosus GG, L. plantarum KCTC 3108, and L. plantarum 200655 at the end of the storage period were 0.78 ± 0.02%, 0.82 ± 0.04%, 0.81 ± 0.06%, and 0.82 ± 0.04%, respectively. This study similarly found that the titratable acidity was higher in yogurts containing bioprotective or probiotic cultures compared to the control yogurt, which contained only the starter cultures.

These findings indicate that bioprotective cultures can effectively control acidity during storage, thereby contributing to the stability of the yogurts. Therefore, analyzing titratable acidity is a crucial parameter in assessing the viability of using bioprotective cultures in fermented dairy products.

3.2. Effect of Bioprotective Cultures on the Inhibition of Molds and Yeasts

It was observed that mold and yeast colonies were virtually absent in all experiments up to 14 days of storage at 7 ± 1 °C (

Table 2. Concentration of molds/yeasts (CFU/mL) as a function of days of storage at 7 ± 1 °C *.

DAYS	C1	C2	H1	F1	H2	F2	H3	F3
1	absent	10	10	absent	absent	absent	absent	absent
7	absent	absent	30	absent	10	10	absent	absent
14	absent	40	10	absent	20	20	absent	absent
21	absent	5440	10	absent	2300	4470	340	absent
28	absent	21,000	absent	absent	2770	18,000	absent	absent

* Note: C1: 0.03% chemical preservative potassium sorbate; C2: 0% chemical preservative and commercial biopreservative cultures; H1: 0.015% chemical preservative and 0.1% HOLDBAC YM-B; F1: 0.015% chemical preservative and 0.1% FRESHQ-11; H2: 0.1% HOLDBAC YM-B; F2: 0.1% FRESHQ-11; H3: 0.2% HOLDBAC YM-B; F3: 0.2% FRESHQ-11.

). In control yogurt C1, which contained only the chemical preservative potassium sorbate at the maximum concentration of 0.03%, no mold or yeast formation was detected throughout the 28-day storage period at 7 ± 1 °C. However, in control yogurt C2, which lacked both chemical preservatives and bioprotective cultures, a significant increase in mold and yeast counts was observed beginning at 21 days of storage, reaching 5440 CFU/mL at 21 days and escalating to 21,000 CFU/mL by 28 days.

In the case of yogurts containing bioprotective cultures combined with half the maximum dosage (0.015%) of the preservative potassium sorbate (experiments H1 and F1), it was observed that only yogurt H1, which used a dosage of 0.1% v/v of commercial culture H, did not achieve complete fungal inhibition, except at 28 days of storage when total fungal inhibition was observed. Conversely, yogurt F1, with a dosage of 0.1% v/v of commercial culture F and 0.015% potassium sorbate, achieved complete fungal inhibition throughout the 28-day storage period, similar to the results observed in control yogurt C1.

However, when the chemical preservative was entirely removed while maintaining the same dosage of 0.1% v/v of bioprotective cultures (experiments H2 and F2), a noticeable increase in mold and yeast formation occurred after 21 days of storage at 7 ± 1 °C, with counts reaching 2300 CFU/mL and 4470 CFU/mL, respectively. By 28 days, mold and yeast counts had further increased, with 2770 CFU/mL for yogurt H2 and 18,000 CFU/mL for yogurt F2. When the dosage of bioprotective cultures was doubled to 0.2% v/v without adding chemical preservatives, yogurt F3, containing commercial culture F, exhibited total inhibition of molds and yeasts throughout the 28-day storage period. Similarly, yogurt H3, with a doubled dosage of commercial culture H, showed no colony formation within 28 days.

Several similar studies have reached comparable conclusions using other bioprotective cultures and have sought to elucidate the mechanisms by which these cultures inhibit mold and yeast growth. For instance, Siedler et al. [34] found that the depletion of the essential trace element manganese by Lactobacillus paracasei and L. rhamnosus was a key mechanism for inhibiting yeast and mold growth that typically deteriorates yogurt. They reported that the inhibition or reduction of spoilage yeasts and molds is linked to the absorption of a significant amount of manganese by bioprotective cultures in dairy products. Thus, the inhibition of mold growth in yogurt and the ability of manganese to restore mold growth appear to be closely associated with the capacity of LAB to efficiently deplete manganese.

Similarly, Shi and Knøchel [35] demonstrated that in yogurt, the depletion of manganese by Lactobacillus plantarum LP37 played a crucial role in inhibiting the growth of Penicillium and Mucor strains. Peng et al. [36] observed that the antifungal activity of Lactobacillus plantarum LPP703 CFS against Penicillium sp. was reduced by pH neutralization, likely due to the organic acids present when L. plantarum LPP703 CFS was added. According to these authors, when the pH is lower than the pKa of the organic acids, the undissociated form predominates and can pass through the fungal cell membrane via passive diffusion, leading to the accumulation of organic acids in the cytoplasm and the inhibition of fungal growth. Conversely, at higher pH levels, organic acids dissociate, and these dissociated acids cannot easily penetrate the cell membrane, thereby reducing the antifungal effect of organic acids [37].

3.3. Effect of Bioprotective Cultures on Yogurt Water Retention Capacity and Syneresis

According to Figure 3, whey separation (syneresis) in all experiments significantly decreased over the storage time (p < 0.05) during the 28 days at 7 ± 1 °C. This indicates that water retention capacity (WRC) significantly increased (p < 0.05) as a function of storage time or shelf life.

Yogurts containing bioprotective cultures exhibited significantly lower syneresis compared to C2 yogurt (p < 0.05) and showed no significant variation (p > 0.05) when compared to C1 yogurt, particularly towards the end of the storage period (28 days). As a result, the WRC was generally higher (p < 0.05) in yogurts with added bioprotective cultures compared to those containing only the initial starter cultures (C2). At the end of the 28-day storage period, yogurts containing bioprotective culture H (H2 and H3) exhibited significantly lower syneresis (p < 0.05) than those containing commercial culture F (F2 and F3).

It was observed that doubling the dosage of bioprotective culture F to 0.2% v/v (yogurt F3) resulted in a significant increase in syneresis (p < 0.05) during storage compared to yogurts using the same culture at a dosage of 0.1% v/v (yogurts F1 and F2). Conversely, when the dosage of culture H was doubled to 0.2% v/v (yogurt H3), there was a decrease in syneresis during most of the storage period compared to yogurts using culture H at 0.1% v/v (yogurts H1 and H2). However, these differences were not statistically significant at the end of the 28-day storage period (p > 0.05).

It is widely recognized that L. rhamnosus and P. freudenreichii are species known for their ability to produce exopolysaccharides (EPS), which contribute to the textural and rheological properties of fermented dairy products such as yogurt. While EPS production can vary among different strains, the literature provides substantial evidence that strains of L. rhamnosus and P. freudenreichii are capable of producing significant amounts of EPS [38,39,40,41,42].

In this study, we utilized commercial bioprotective cultures HOLDBAC YM-B (a combination of L. rhamnosus and P. freudenreichii subsp. shermanii) and FRESHQ-11 (L. rhamnosus) to assess their impact on yogurt quality. Although the specific EPS production capabilities of these commercial strains have not been characterized by the manufacturers, their antifungal properties have been well documented, as this is their primary intended use. These bioprotective cultures are typically employed alongside starter cultures such as Streptococcus salivarius subsp. thermophilus, a species well known for its robust EPS production and its role in enhancing yogurt viscosity.

Given the established EPS production capabilities of the species involved, it is reasonable to hypothesize that the observed improvements in yogurt texture and viscosity may be partially attributed to EPS production by these bioprotective cultures, either directly or through synergistic effects with the starter cultures. While direct quantification of EPS was not conducted in this study, the existing body of literature supports the likelihood of EPS production by the strains used, and no studies have been found to suggest otherwise. Further research could involve targeted analysis of EPS production to confirm these effects in the specific strains used in this study.

The study by Zhao and Liang [17] reported similar results, concluding that the WRC values of yogurt samples containing the bioprotective culture L. plantarum were significantly higher than those of the control (p < 0.05) during storage. These findings suggest that the addition of L. plantarum MC5 likely improved the syneresis characteristics of yogurt during storage. It has also been reported that milk fermented with EPS-producing bioprotective cultures exhibits enhanced water retention capacity [43].

The water-binding capacity of EPS limits whey separation in yogurt and stabilizes the protein gel structure, resulting in greater cohesion within the yogurt samples. The water-binding capacity of EPS-producing LAB is influenced by factors such as the type, quantity, and distribution of EPS, the interaction between protein networks and EPS, and the duration of yogurt fermentation [44].

According to Ziarno et al. [43], WRC values are influenced by the microbial composition of the starter culture, likely due to the acidifying properties of the bacteria used. In their study, lower WRC values were recorded for samples fermented exclusively with propionic bacteria (PAB) cultures, while a mixture of initial yogurt bacteria and propionic cultures produced WRC values comparable to those found in samples fermented solely with initial yogurt cultures.

Furthermore, in the study by Fayyaz et al. [18], for all yogurts with LAB added, WRC significantly increased over time (p < 0.05), while for the control yogurt sample, the change was not significant (p > 0.05). These results are similar to those found in the present research, and according to the authors, interactions occur during storage periods in terms of acid production due to culture growth, causing casein particles to retain water molecules more efficiently, resulting in decreased syneresis [45]. The increase in protein WRC due to pH decrease parallel to LAB growth leads to increased yogurt curd stability [45].

3.4. Effect of Bioprotective Cultures on Yogurt’s Apparent Viscosity

Figure 4 presents the results for apparent viscosity at a shear rate of 35 s⁻¹ during the shelf life period. The apparent viscosity of all yogurt samples exhibited notable fluctuations throughout the storage period, indicating complex biochemical and microbial interactions within the yogurt matrix. Initially, the C1 samples presented higher viscosity values than C2, suggesting a variance in the matrix’s stability or response to the storage conditions. Interestingly, by Day 7, C2 samples surpassed C1 in viscosity, highlighting a potential difference in the stabilization or breakdown processes occurring within these samples.

Samples inoculated with bioprotective cultures (H1, H2, H3, F1, F2, F3) demonstrated significant variations in viscosity, underscoring the profound impact of these cultures on yogurt’s rheological properties. The H series, in particular, showed a marked increase in viscosity from Day 1 to Day 7, with H2 reaching the highest viscosity among all tested samples. This trend suggests that the synergistic effect of L. rhamnosus and P. freudenreichii subsp. shermanii within the H series may contribute to enhanced network formation or stability within the yogurt matrix, potentially through the production of EPS or other metabolites that influence viscosity.

Conversely, the F series samples exhibited a general decrease in viscosity after Day 7, with notable differences between samples. F2, for instance, showed a dramatic reduction in viscosity, indicating a distinct interaction of F cultures with the yogurt matrix that differs from that of the H cultures. F3 presented an initial increase followed by a significant decrease in viscosity by Day 28, suggesting an early stabilizing effect by the bioprotective culture that diminishes over time.

The production of EPS can substantially explain the marked variance in viscosity among samples treated with bioprotective cultures. These high-molecular-weight polysaccharides, secreted by specific LAB strains during fermentation, are known to augment yogurt’s viscosity by enhancing water retention and contributing to a more gel-like structure [46]. The H series, particularly H2, exhibited a pronounced increase in viscosity early in the storage period, which we attribute to EPS production. This suggests that the synergistic effects of L. rhamnosus and P. freudenreichii subsp. shermanii are conducive to a robust EPS-mediated increase in the matrix’s resistance to flow [38].

EPS not only hold water but also interact with milk proteins to strengthen and stabilize the gel network, thereby augmenting the yogurt’s overall viscosity [47]. The interaction between EPS and the milk protein network potentially explains the observed rheological behavior of the H series samples, which displayed a consistently higher viscosity, suggesting a more cohesive and stable gel formation within the yogurt matrix.

Beyond EPS, other metabolites LAB produce, such as organic acids and proteolytic enzymes, play crucial roles in modulating the yogurt’s texture [48]. Organic acids contribute to casein micelle aggregation and gel formation by lowering the milk’s pH, whereas proteolytic enzymes alter the protein network by hydrolyzing milk proteins. These biochemical processes are instrumental in defining the final textural and sensory attributes of yogurt.

3.5. Effect of Bioprotective Cultures on Yogurt Texture

Yogurt is a mixture of biopolymers such as proteins, polysaccharides, and fats. The microstructure of milk protein gels and their rheological properties can affect texture, sensory properties, fat globules, and storage stability [49]. The yogurt microstructure is formed by a three-dimensional network of casein micelle aggregates, where the globular shape is observable and interspersed with void zones [50].

According to

Table 3. Yogurt texture properties during shelf life: firmness (g-force), consistency (g-sec), cohesiveness (g-force), and viscosity index (g-sec) *.

	Firmness (g-force)
Days	C1	C2	H1	F1	H2	F2	H3	F3
1	22.8 ± 0.6 bBC	24.0 ± 0.2 aAB	22.8 ± 0.2 cBC	21.9 ± 0.4 cCD	22.9 ± 0.7 bBC	21.2 ± 0.2 cD	22.6 ± 0.4 bCD	24.7 ± 0.4 bA
7	25.1 ± 0.7 aB	24.5 ± 0.6 aB	24.4 ± 0.7 bB	23.0 ± 0.6 bcCD	23.9 ± 0.6 abBC	22.2 ± 0.5 bcD	22.1 ± 0.1 bD	26.9 ± 0.5 aA
14	25.2 ± 0.3 aAB	24.8 ± 0.5 aAB	26.1 ± 0.5 aA	24.7 ± 0.7 aAB	24.2 ± 0.3 aBC	23.2 ± 0.4 bC	23.3 ± 0.3 abC	25.1 ± 0.4 bAB
21	25.3 ± 0.4 aAB	24.7 ± 0.4 aABC	26.0 ± 0.5 aA	23.9 ± 0.3 abC	24.3 ± 0.3 aBC	24.7 ± 1.7 aABC	24.2 ± 0.3 aBC	25.2 ± 0.5 bABC
28	25.7 ± 0.4 aA	24.7 ± 0.5 aAB	25.2 ± 0.5 abAB	24.0 ± 0.7 abBC	23.9 ± 1.2 abBC	22.6 ± 0.4 bC	22.7 ± 0.2 bC	25.1 ± 0.4 bAB
	Consistency (g-sec)
1	475.1 ± 7.2 cCD	499.4 ± 7.0 bAB	474.5 ± 10.3 cCD	460.5 ± 3.8 cDE	485.6 ± 11.7 cBC	449.3 ± 3.8 cE	461.3 ± 10.9 dDE	518.0 ± 11.5 cA
7	526.9 ± 11.6 bB	516.8 ± 5.0 abB	522.0 ± 7.3 bB	492.7 ± 6.3 bCD	505.7 ± 4.7 abBC	475.1 ± 7.4 abDE	463.8 ± 5.4 cdE	575.6 ± 14.0 aA
14	541.5 ± 10.2 abAB	531.3 ± 3.8 aBC	560.8 ± 13.4 aA	524.4 ± 6.7 aBC	512.4 ± 5.5 aC	486.6 ± 4.8 abD	488.3 ± 7.3 bD	537.6 ± 5.8 bB
21	553.0 ± 9.3 aA	523.4 ± 9.1 aB	559.1 ± 8.5 aA	520.2 ± 10.9 aB	511.3 ± 8.5 abBC	491.3 ± 14.5 aC	510.9 ± 8.4 aBC	545.2 ± 11.0 bA
28	549.2 ± 9.9 aA	527.4 ± 6.4 aBC	559.6 ± 2.6 aA	511.6 ± 3.2 abCD	492.9 ± 12.1 bcDE	470.3 ± 8.9 bF	480.8 ± 4.2 bcEF	541.3 ± 7.9 bAB
	Cohesiveness (g-force)
1	16.8 ± 0.4 cAB	17.5 ± 0.7 abA	16.9 ± 0.3 cAB	16.1 ± 0.1 cBC	16.6±0.6 bABC	15.5± 0.7 bC	17.0 ± 0.6 aAB	17.3 ± 0.4 bA
7	17.5 ± 0.7 bcBC	17.4 ± 0.2 bBCD	17.9 ± 0.8 bcAB	16.3 ± 0.2 bcD	17.2 ± 0.3 abBCD	16.5 ± 0.6 abCD	16.8 ± 0.2 aBCD	19.0 ± 0.5 aA
14	18.1 ± 0.5 abAB	17.7 ± 0.7 abB	19.0 ± 0.2 aA	17.5 ± 0.5 aBC	17.8 ± 0.4 aAB	16.5 ± 0.3 aC	17.2 ± 0.7 aBC	18.2 ± 0.3 abAB
21	19.0 ± 0.5 aA	18.5 ± 0.7 aAB	18.5 ± 0.4 abAB	16.8 ± 0.5 abcC	17.4 ± 0.3 abBC	16.7 ± 0.4 aC	17.6 ± 0.4 aBC	18.0 ± 0.5 abAB
28	18.3 ± 0.3 abAB	18.1 ± 0.7 abAB	18.8 ± 0.4 abA	17.1 ± 0.1 abBC	17.4 ± 0.4 abBC	16.3 ± 0.2 abC	17.6 ± 0.7 aB	18.0 ± 0.6 abAB
	Viscosity index (g-sec)
1	6.5 ± 0.8 cAB	7.6 ± 0.8 aA	5.7 ± 0.6 cABC	3.8 ± 0.5 bC	5.9 ± 0.6 aABC	4.5 ± 0.2 aBC	5.5 ± 0.9 abABC	6.7 ± 1.0 cAB
7	9.6 ± 0.9 bB	7.6 ± 0.8 aBCD	8.1 ± 1.0 bBC	5.8 ± 1.6 abDE	6.7 ± 1.2 aCDE	4.5 ± 0.5 aE	4.5 ± 0.3 bE	12.4 ± 1.1 aA
14	11.6 ± 0.5 abA	8.0 ± 1.6 aB	13.2 ± 1.3 aA	7.3 ± 0.2 aBC	7.7 ±0.7 aB	5.0 ± 0.8 aC	5.3 ± 0.5 bC	9.1 ± 0.6 bB
21	12.5 ± 1.3 aA	8.9 ± 0.9 aB	12.9 ± 1.3 aA	6.8 ± 0.1 aBC	7.4 ± 0.6 aBC	5.6 ± 0.6 aC	7.5 ± 0.7 aBC	7.6 ± 1.4 bcBC
28	10.6 ± 1.9 abB	8.4 ± 1.3 aBCD	13.5 ± 0.2 aA	7.7 ± 1.5 aCDE	6.2 ± 1.3 aDE	3.6 ± 0.7 aF	6.0 ± 0.6 abE	9.3 ± 1.5 bBC

* Note: a, b, c—Significant differences between the means of each line (p < 0.05). A, B, C, D, E, F—Significant differences between the means of each column (p < 0.05).

, yogurt texture parameters (firmness, consistency, cohesiveness, and viscosity index) significantly increased during storage at 7 ± 1 °C (p < 0.05), except for C2, H3, and F3 yogurts, which showed no significant variation in these parameters (p > 0.05) at 28 days. In experiments containing commercial bioprotective cultures, texture parameters were significantly lower (p < 0.05) than in C1 and C2 yogurts during the storage period, except for F3 (F culture at 0.2% v/v dosage) and H1 (H culture at 0.1% v/v dosage) yogurts.

It can also be observed in Table 3 that when the dosage of the F bioprotective culture was doubled to 0.2% v/v (yogurt F3), there were significant increases in firmness, consistency, cohesiveness, and viscosity index (p < 0.05) during storage time compared to yogurts using the same culture at a dosage of 0.1% v/v (yogurts F1 and F2). The opposite behavior was observed when the dosage of the culture was doubled to 0.2% v/v (yogurt H3); i.e., there was a significant decrease in these parameters (p < 0.05) during storage time compared to yogurts using the same culture at a dosage of 0.1% v/v (yogurts H1 and H2), with the exception of cohesiveness and viscosity index from Day 21 onwards.

These results are in accordance with the study by Zhao and Liang [17], who observed that the firmness of yogurts containing the bioprotective culture L. Plantarum MC5 was lower than that of control yogurt S. Therefore, the addition of L. Plantarum MC5 significantly reduced the firmness of yogurt samples. According to these authors, similar results were reported by Bancalari et al. [51], where it was observed that the firmness of yogurts made with EPS-producing cultures was generally lower than that of a control yogurt made without initial cultures producing EPS. EPS can affect the texture properties of a yogurt clot by reducing its firmness [52].

In the study by Zhao and Liang [17], the firmness of samples from control yogurt S also gradually increased during storage time, while those with L. plantarum MC5 remained reasonably constant, which did not occur in the present study with other bioprotective cultures. According to the authors, this observation may be due to the difference in EPS production and post-acidification of yogurt during storage. Yildiz and Ozcan [53] also reported that firmness and consistency were affected by syneresis, pH decrease, and increased casein hydration in yogurts with long storage times. The study by Zhao and Liang [17] also found that the interaction between the proportion of L. plantarum MC5 addition and storage time of yogurt samples had a significant effect on yogurt firmness, consistency, and cohesiveness (p < 0.01).

In the study by Ziarno et al. [43], an increase in yogurt hardness or firmness during refrigeration was also found in almost all stored samples; these changes were already statistically significant after 7 or 14 days of refrigerated storage. The magnitude of the changes depended on the bacterial culture used to ferment the yogurt. In this study, the greatest changes were observed when a propionic acid bacterium (PAB) starter was used, where a decrease in hardness compared to control yogurt was observed only with the initial cultures. This result is in line with that found in the present research, where it was observed that increasing the dosage of culture H containing the propionic bacterium (PAB) to 0.2% v/v further decreased the firmness, consistency, cohesiveness, and viscosity index of yogurt compared to control yogurts.

The texture (both hardness and adhesiveness) of yogurt products depends on the composition of the raw material, the type of bacterial culture used, the fermentation method, and texturizing additives, among other factors [54]. In contrast, Vinderola et al. [55] found that the rheological properties of fermented dairy products depend on titratable acidity (higher titratable acidity correlates with greater hardness). The composition of proteins present in yogurt is extremely important [56]. The acidifying properties of propionic bacteria, which are weaker than those of yogurt starter culture, did not improve the texture characteristics of these yogurt samples. During storage of yogurt samples, a statistically significant increase in adhesiveness was observed, except for yogurts with propionic culture added, whose adhesiveness decreased by 21 days. The acidifying properties of propionic bacteria, which are weaker than those of starter cultures, did not improve the adhesion values of yogurt samples [43]. The results of this study are consistent with the present research; as previously reported, the cohesiveness of yogurts also showed a trend to increase with storage days at 7 ± 1 °C.

4. Conclusions

In conclusion, this study highlights the potential of commercial bioprotective cultures as effective natural alternatives to chemical preservatives in yogurt production. The addition of these cultures led to a significant increase in titratable acidity (%) compared to the control yogurt (C2), which contained only starter cultures without any preservatives. This increase in acidity, coupled with a progressive rise during storage, underscores the active role of bioprotective cultures in enhancing the yogurt’s safety by creating an environment less favorable to spoilage organisms.

Particularly noteworthy is the fungal inhibition achieved with a 0.2% v/v dosage of bioprotective cultures, even without potassium sorbate. The results demonstrated that these cultures, especially L. rhamnosus (commercial culture F), performed comparably to the chemical preservative control (C1) in preventing fungal growth over the 28-day storage period. This finding is promising for future applications at both pilot and industrial scales, suggesting that bioprotective cultures could effectively replace or reduce the reliance on chemical preservatives in yogurt. Additionally, this study observed a reduction in syneresis across all experiments, with texture parameters improving over time. Although the use of bioprotective cultures resulted in lower values for firmness, consistency, cohesiveness, and viscosity index compared to C1 and C2, these differences are not necessarily detrimental. It is important to consider that no thickening agents or powdered milk were used in this study, commonly added to enhance texture and reduce syneresis in commercial yogurt products.

Overall, incorporating bioprotective cultures in yogurt production offers a promising avenue for improving product safety and shelf life while meeting consumer demand for cleaner labels. Future research and formulation adjustments could further optimize the texture and sensory properties, making these natural bioprotective agents a viable option for large-scale yogurt manufacturing.