1. Introduction
Yogurt is a fermented dairy product consumed worldwide. Product acceptability is largely dependent on its texture and flavour attributes, while preferences may vary widely among countries and regions. Aldehydes, alcohols, and acids are typical volatile compounds that contribute to the flavour development of yogurt, which are either already present in milk or produced by the bacterial culture [1,2,3]. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) are predominantly used in traditional set-type yogurt cultures, due to the protocooperation of species during fermentation [2]. L. bulgaricus breaks down casein to produce free amino acids and peptides to aid the growth of S. thermophilus, which in return produces formic acid and carbon dioxide to help the growth of L. bulgaricus [4]. This exchange of metabolites promotes the growth of each species, leading to a faster decrease in pH during yogurt production [5,6].
Before yogurt production, milk is standardized, pasteurized, and treated with heat, which removes most of the CO2 naturally present in the milk [7,8]. L. bulgaricus relies on S. thermophilus, a producer of CO2, as a by-product of metabolic reactions. CO2 and formic acid are a precursor for nucleotide synthesis [8]. L. bulgaricus produces cell-envelope proteinases (CEP) to hydrolyze large proteins in milk, such as casein [3,9]. The CEP is an enzyme that initiates the process of proteolysis by producing di-, tri-, and oligopeptides, which are transported into the cell for further processing by intracellular peptidases [2,10]. Peptides are further hydrolyzed by peptidases into free amino acids, which are involved in metabolic regulation and protein biosynthesis. This enhanced proteolysis to peptides and amino acids promotes growth and lactose fermentation, leading to more rapid lactic acid production and acidification during fermentation [11]. Only one CEP, the PrtB, has been identified in L. bulgaricus [12]. S. thermophilus has three distinct surface proteinases, namely PrtS, HtrA, and SepM [13]. Of these, PrtS is important for acidification and is involved in the flavour development of yogurt [10,12,14]. It is important to note that the presence of PrtB and PrtS in L. bulgaricus and S. thermophilus is strain-dependent; thus, not every strain exhibits cell envelope proteinase activity [10,12,15,16].
Free amino acids can be converted into volatile compounds such as amines, aldehydes, phenols, and alcohols that contribute to flavour [2,17]. Furthermore, the oxidation–reduction potential (Eh) measures the tendency of a biological system to gain or lose electrons, reflecting the overall oxidative or reductive capacity. In the milk environment, LAB modulate the redox potential by donating or accepting electrons during key metabolic processes, which directly affects flavour formation [18]. For example, the reduction of pyruvate into lactic acid, a key reaction in glycolysis, involves an electron transfer that lowers the redox potential, leading to acidification and the development of a sour taste. Additionally, LAB can convert citrate into diacetyl and acetoin; this requires redox reactions, altering the oxidative balance and contributing to buttery notes. The oxidation of sulfur-containing amino acids, such as methionine, also involves redox changes, producing volatile sulfur compounds [19,20]. These redox-sensitive reactions with LAB can shape the overall sensory profile of yogurt. Depending on the medium used and the environment, oxygen and nitrogen have been found to alter the metabolic regulation of specific pathways depending on the oxidation–reduction potential [21,22]. Martin et al. [21] found that more sulphides were produced in the presence of oxygen under reducing conditions, whereas higher amounts of acetaldehyde were produced under oxidizing conditions in low-fat yogurt.
An inconsistent fermentation performance due to variations in metabolic processes can impact product quality [23]. For instance, the differential expression of key genes involved in glycolysis and fermentation, such as those encoding for pyruvate kinase and lactate dehydrogenase, can lead to variations in metabolic flux and ultimately affect fermentation outcomes [24]. Characterizing the phenotypic profiles of individual isolates enables the combination of strains in configurations that could enhance the consistency of aroma and flavour production. Studies have demonstrated significant variability in the acidification and proteolytic activities of S. thermophilus and L. bulgaricus, highlighting a broad spectrum of phenotypic responses [10,15]. Multivariate statistical analysis has effectively integrated multiple phenotypic variables, such as volatile compounds and acidification parameters, to reveal unique phenotypic signatures among various Lactococcus lactis strains [25], but this approach has not yet been applied to S. thermophilus and L. bulgaricus. By examining the interactions and correlations among these diverse traits, researchers can identify the factors influencing the fermentation performance and sensory outcomes. Conducting such studies could uncover phenotypic patterns and compatibility insights relevant to optimizing mixed fermentation processes. The current study analyzed 10 industrially relevant phenotypic traits of yogurt starter bacteria, their aroma-generating properties in milk and the compatibility between S. thermophilus and L. bulgaricus isolates.
2. Materials and Methods
2.1. Isolate Growth Conditions, Cultivation, Collection and Identification
Bacteria were isolated from three starters; Starter 1 (S1) has been previously used in yogurt production while Starter 2 (S2) and Starter 3 (S3) are routinely used in commercial yogurt production. One millilitre of each starter sample was homogenized in 9 mL of 0.1% peptone water, serially diluted, and plated on De Man, Rogosa and Sharpe (MRS) agar (Oxoid Microbiology, Nepean, ON, Canada) for the enumeration of L. bulgaricus and M17 agar (Oxoid Microbiology, Nepean, ON, Canada) for the enumeration of S. thermophilus and incubated for 48 h at 42 °C [26]. Twenty-four isolates (12 from MRS and 12 from M17) were collected for each starter culture (a total of 72 isolates from 3 starters). Colonies were streaked twice in series on the appropriate medium for purification and stored in glycerol at −80 °C. During this isolation procedure, each colony was verified for ropiness by stretching it with the inoculating loop and observing a filament. For activity tests, 10 mL of overnight culture incubated at 37 °C (MRS broth for L. bulgaricus and M17 broth for S. thermophilus) was centrifuged at 10,000× g for 10 min at 25 °C. The pellets were collected and transferred to 10 mL of skim milk, which were incubated overnight at 37 °C. After 16 h of incubation, the aroma of each fermented milk sample was detected by trained lab personnel by wafting the headspace air towards the nose and identified as the following: butter, intense butter, sour, cooked milk or other (off-odor). Seventy-two isolates were identified to the species level using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis.
2.2. Acidification Activity
Frozen cultures were inoculated in 10 mL of skim milk (S. thermophilus) or 10 mL pf skim milk supplemented with 1 mM of sodium formate [4] (Fisher Scientific, Mississauga, ON, Canada) (L. bulgaricus) and incubated overnight at 37 °C. Subcultures of each monoculture in their respective media were used to inoculate 100 mL of 3.25% microfiltered milk at 3% w/v and incubated for 16 h at 40 °C to mimic commercial yogurt production parameters. The acidification capacities of all cultures were monitored using an iCinac System (AMS Alliance, Frépillon, France) [25], providing the pH and oxidation–reduction potential (ORP) every minute for up to 16 h. The following parameters were calculated: time to pH 4.6 (tpH4.6), maximum acidification rate (Vmax), maximum redox potential (Eh7min), time required to reach maximum redox potential (tmin), maximum difference between two measures (Δmax) and the time required to reach maximum difference (t*). After 1 day and 7 days of fermentation, the titratable acidity was measured using an Autotitrator T50 (Mettler Toledo, Columbus, OH, USA).
2.3. Proteolytic Activity
Fast–Slow Difference Agar (FSDA) was used to differentiate between the Prt+ and Prt− phenotypes of all bacterial isolates collected from each starter culture [10]. The protease-positive (Prt+) phenotype appears as yellow, with opaque colonies surrounded by yellow zones, whereas protease-negative (Prt−) isolates appear small, flat, and translucent on FSDA agar. The proteolytic potential of thirty-six Prt+ isolates from the three original starter cultures was quantified using the o-phthaldialdehyde (OPA) method, following that of Savoy De Giori and Hebert [26] with some modifications [27]. The cells from overnight cultures were collected by centrifugation for 10 min at 4 °C and washed twice with phosphate buffer (pH 7.4). The cell pellet was resuspended in 10 mL of phosphate buffer, and the suspension was inoculated into 10 mL of reconstituted skim milk (RSM) at a 1% concentration. Each sample was vortexed and incubated for 12 h at 30 °C. After 12 h, the samples were vortexed until homogenized, and 250 μL was thoroughly mixed with 50 μL of dH2O and 500 μL of 0.75 N trichloroacetic acid (TCA) (Sigma-Aldrich, Oakville, ON, Canada). The coagulated milk was vigorously vortexed, allowed to stand for 15 min, and then centrifuged at 13,000× g for 15 min. Ten microliters of the supernatant was transferred to a 96-well flat-bottom plate, and 200 μL of OPA reagent was added to each well. Each sample was tested in triplicate using a spectrophotometer at 340 nm against blank uninoculated milk. The millimoles per millilitre of α-amino acid produced were calculated using the following equation:
where is the observed change in absorbance at 340 nm, F is the dilution factor corresponding to the assay procedure, and is the molar absorption coefficient (6000/M−/cm).2.4. CEP Activity Test
The procedure used for the CEP activity test followed the method described in Stefanovic et al. [3], with some modifications. After the overnight incubation of milk cultures, cells were centrifuged at 4000× g for 5 min at 25 °C. The fat layer was removed, and the cells were washed three times with 500 µL of 50 mmol/L Tris-HCl buffer and 500 µL of 2 mmol/L CaCl2. After a third wash, the supernatant was discarded. The EnzCheck® Green Fluorescence E-6638 kit (Thermo Fisher Scientific, Waltham, MA, USA) was used, and the reagents were prepared according to the manufacturer’s instructions. A digestive buffer (500 µL) was then added to resuspend the cells. One hundred microliters of cell suspension and 100 µL of BODIPY®FL casein solution for each sample were placed in a 96-well microplate in triplicate, which was incubated in the dark at 37 °C for 24 h. Fluorescence readings were obtained using a Biotek BioSynergy Reader (Ex/Em 505/513 nm, Highland Park, Winooski, VT, USA). The results are reported as direct fluorescence readings.
2.5. Growth Inhibition Assay
The growth inhibition of isolates from the 2 starter cultures (S2 and S3) that are still routinely used in production was tested. A total of 24 S. thermophilus isolates and 24 L. bulgaricus isolates were tested against each other using the soft agar overlay technique with some modifications by Hockett and Baltrus [28]. The S. thermophilus and L. bulgaricus isolates were grown in 10 mL of buffered M17 broth and 10 mL of buffered MRS broth, respectively, at 37 °C. A lawn of overnight culture for each isolate was then plated on buffered M17 agar or buffered MRS agar and grown aerobically and anaerobically, respectively, at 37 °C for 24 h. The top of each isolate lawn was sectioned into 6 parts and then inoculated with a 5 µL drop of an overnight liquid culture of each isolate. The drops were left to dry and then incubated aerobically at 37 °C for S. thermophilus isolates and anaerobically at 37 °C for L. bulgaricus. After 24 h, the plates were inspected for the presence of any clear zones (inhibition zones) around the isolate. Isolates that produced a zone were classified as inhibitory against the target isolate.
2.6. Data Analysis
Each phenotypic test was performed in triplicate experiments (not technical replicates of the same culture), and the means for each were used for further analysis. Eight phenotypic measures were normalized by centering (subtracting the population mean) and unit variance scaling (dividing the population mean difference by the population standard deviation) for comparison. The 10 variables (tpH4.6, Vmax, Eh7min, Δmax, t*, tmin, proteolytic activity, CEP activity, titratable acidity (24 h) and titratable acidity (after 7 days)) were clustered for the 36 S. thermophilus isolates and 36 L. bulgaricus isolates by the Euclidian distance metric and Ward criterion using agglomerative hierarchical clustering (AHC) [25]. The hierarchical groups of mutual subsets in which maximally similar isolates were determined were based on all variables. To investigate the relationship between clusters and the acidification capacity of isolates, an analysis of variance (ANOVA) was performed. The significance level was set to a p-value cut-off of 0.05 using the Dunn–Sidak method for all pairwise comparisons between variables and clusters. Non-metric multidimensional scaling (NMDS) was used to plot the Euclidian distance and Ward criterion of sample comparisons.
3. Results
3.1. Phenotypic Diversity
The milk used to evaluate each activity parameter (redox potential, acidification activity, proteolytic activity, and CEP activity) of the isolates had an average pH of 6.6 ± 0.4 at 40 °C, and the average initial Eh7min value of the inoculated milk was 191.5 ± 12 mV.
3.1.1. S. thermophilus
Each cluster of S. thermophilus consisted of fast and slow acidifying isolates (Figure 1a). Most isolates belonged to clusters ST2 and ST3 and each cluster had isolates that represented all three starter cultures (Figure 2a). All clusters contained isolates showing a mix of aroma features from the 10 mL milk cultures. Cluster ST2 showed the greatest variation among isolates grouped together with all aroma traits: sour, butter, intense butter, and cooked milk. ST4 was the only cluster that did not have an isolate with a sour aroma, and ST5 did not have an isolate with a cooked milk aroma. Cluster ST1 included five isolates with a ropy phenotype.
The redox potential of the milk after 16 h of fermentation ranged from 114 mV to −176 mV, with only 9 out of 36 isolates having low reduction activity. S. thermophilus with higher reducing activity were grouped into clusters ST2, ST3, and ST4, with the ST2 cluster having the highest reducing activity (Eh7min = −99.2 ± 29.6 mV). However, the isolates in cluster ST4 had a high reduction power Eh7min (−80.8 ± 38.4 mV) and were the fastest to reach Δmax (Table 1). The isolates clustered in ST1 displayed a low reduction power (38.6 ± 28.8 mV), resulting in the slowest reaching of Δmax (t* = 9.99 ± 1.91 h). A significant difference in the average reducing activity was seen between ST1 and ST2, ST3 and ST4 (p < 0.05). The time needed to reach a pH of 4.6 ranged from 6 h to 16 h (Figure 1b). All clusters had similar averages regarding the time needed to reach a pH of 4.6, with clusters ST3 taking the longest time (12.3 ± 1.42 h) and ST1 having the shortest time (11.1 ± 2.66 h). The Vmax reflected the results of the time needed to reach a pH of 4.6, where ST1 had the fastest average acidification rate but ST2 has the slowest average acidification rate. No significant difference was seen between any clusters regarding the time needed to reach 4.6 and Vmax, with p > 0.05. The proteolytic activity ranged from 19 millimoles/mL to 139 millimoles/mL. ST4 had the highest average proteolytic activity, whereas ST2 had the lowest (Table 1). The CEP activity ranged from 239.5 to 450.3 direct fluorescence units amongst all the isolates. Consistent with proteolytic activity, the ST4 isolates had the highest average CEP activity (430.7 ± 20.3). A significant difference in CEP activity was seen between the clusters, with ST4 isolates having a significantly different CEP activity (p < 0.05) compared to ST1, ST2 and ST3, where ST1 showed the lowest average CEP activity (289 ± 18.7).
3.1.2. Lactobacillus delbrueckii subsp. bulgaricus
Each cluster of L. bulgaricus consisted of fast and slow acidifying isolates (Figure 1b; Table 2). Each cluster consisted of a mix of isolates from all three starters except for LB3. The majority of the isolates in cluster LB1 were from S3 and those in LB4 were from S1. All clusters but LB3 contained isolates showing a mix of aroma features from the 10 mL fermented milk cultures (Figure 2b). The sour aroma was found in all clusters but LBP3, and isolates with an intense butter aroma were only in clusters LB4 and LB5 (Figure 2b). Cluster LB5 showed the greatest variation among the isolates grouped together with all aroma traits: sour, butter, intense butter, cooked milk and off-aroma. LB2 and LB5 consisted of isolates that did not have typical yogurt fermentation sensory properties, such as a stale smell (Supplementary Material Table S2). All the isolates from cluster LB1 (n = 11) and eight out of nine isolates from cluster LB2 showed a ropy phenotype.
A total of five clusters were constructed based on 10 phenotypic measures for L. bulgaricus, with clusters LB1 and LB2 containing the highest numbers of isolates (Table 2). The redox potential for each isolate ranged from 110 mV to −163 mV, with 11 out of 36 isolates having low reduction activity. These 11 isolates were grouped among clusters LB4 and LB5, and all other clusters contained isolates with high reducing activity. LB1 had the highest mean reducing activity (Eh7min = −197 ± 21.8 mV) but cluster LB3 isolates had higher levels of oxidizing activity (Eh7min = −90.3 ± 22.9 mV), also consisting of isolates with the longest tph4.6. LB2 had the slowest average time needed to reach the Δmax (t* = 5.38 ± 3.89). A significant difference in average reducing activity was seen between clusters LB1 and LB2 with clusters LB3, LB4 and LB5 p < 0.05). The average time needed to reach a pH of 4.6 ranged from 4.8 to 16 h, except for two isolates that did not reach the target pH before 6 h. A significant difference in acidification rate (Vmax) was seen in cluster LB3 compared to the four other clusters. The proteolytic activity ranged from 27 millimoles/mL to 173 millimoles/mL. LB4 had the highest average proteolytic activity, whereas LB1 had the lowest (Table 2). Clusters LB1, LB 4 and LB5 showed significant differences with p < 0.05. The CEP activity ranged from 276 to 756 direct fluorescence units amongst all the isolates. LB3 had the highest average CEP activity (748 ± 10.3). The lowest average CEP activity was observed in LB5 (323 ± 44.8). A significant difference in CEP activity was seen between all the clusters with p < 0.05 (Table 2).
3.2. Compatibility of Isolates
All 24 isolates of S. thermophilus showed no antagonistic behaviour against the other isolates of the same species. Similarly, all 24 isolates of L. bulgaricus showed no antagonistic behaviour against other isolates of the same species. In addition, 67% of S. thermophilus isolates showed antagonistic activity against some L. bulgaricus isolates, regardless of the cluster or starter origin. Three L. bulgaricus isolates exhibited antagonistic behaviour against 54% of the S. thermophilus isolates. A fourth L. bulgaricus isolate showed weak antagonistic behaviour against 3 out of the 24 S. thermophilus isolates. In total, 44% of the antagonistic S. thermophilus and all four antagonistic L. bulgaricus isolates displayed fast acidification.
4. Discussion
The main purpose of this study was to use the statistical comparison of multiple parameters of milk fermentation to determine the phenotypic diversity of S. thermophilus and L. bulgaricus isolates commonly combined into starters used in yogurt production. The clustering of the 72 isolates from three starter culture communities revealed distinct groups with shared phenotypic traits such as acidification activity, redox potential, proteolytic abilities, and titratable acidity, highlighting strain-specific differences that could impact fermentation outcomes. Each cluster in this study represents a phenotypic signature that arises from the specific, mathematically determined combination of phenotypic traits. Despite being derived from starter cultures designed for consistent yogurt production, phenotypic variation can influence microbial interactions such as cross-feeding and competition that can further influence trait expression, allowing researchers to classify new strains by comparing their phenotypic profiles with known clusters. By comparing the phenotypic data of these new strains to the established clusters, researchers can select similar characteristics for formulating mixed cultures.
The glycolytic and proteolytic activities of LAB can influence the oxidation–reduction potential (ORP) during yogurt fermentation by altering the redox balance [22]. Brasca et al. [18] found that within the same species of LAB isolated from cheese, there was variation in ORP, but a correlation between the phenotypic parameters and t* was found, which is the time at which the maximum difference in ORP occurred. They also found that slower acidifying strains had more reducing activity, similar to the findings of this study. At the onset of fermentation, the ORP of milk is relatively high due to dissolved oxygen. S. thermophilus actively depletes oxygen via the NADH oxidase pathway, where NADH donates electrons to oxygen, reducing it to water, while facilitating the regeneration of NAD+ for continued glycolytic activity [29]. L. bulgaricus, being anaerobic, does not directly reduce oxygen but instead contributes to the production of reducing agents such as lactic acid and sulfhydryl compounds (cysteine, glutathione), which further lower the ORP. As a result, the decreasing ORP promotes anaerobic conditions that drive further fermentation processes, including proteolysis and the synthesis of aroma compounds such as diacetyl and acetoin [21]. The observed differences in redox potential between L. bulgaricus and S. thermophilus monocultures can be attributed to the variation in the flux of their metabolic activity. L. bulgaricus primarily employs the Embden–Meyerhof–Parnas (EMP) pathway, leading to lactic acid production and limited reducing equivalents, while S. thermophilus, with its heterofermentative capabilities, can generate additional reducing agents such as ethanol and formate, thereby lowering ORP more effectively [30,31]. Furthermore, the lower NADH oxidase activity in L. bulgaricus compared to S. thermophilus limits its ability to maintain a reduced environment, resulting in higher redox potential and reduced fermentation capacities [32,33,34]. When supplemented with formate, L. bulgaricus may struggle to efficiently regenerate NAD+ under high redox conditions, leading to NADH accumulation and further metabolic limitations that cause more oxidative stress and hinder the overall efficiency of fermentation [35,36,37].
In this study, clusters ST1, ST4, LB4, and LB5 exhibited high proteolytic activity and had a lower ORP, contributing to a more reducing environment. However, strains with excessive proteolytic activity can contribute to bitterness due to the production of bitter peptides, resulting in undesirable flavour characteristics [38]. Moreover, clusters of S. thermophilus and L. bulgaricus that generate more oxidizing conditions (between +50 to +100 mV) have been shown to enhance and stabilize aroma compounds, such as acetaldehyde, during storage [21,37]. However, a reduction in ORP can promote the production and accumulation of sulfur-containing compounds, such as hydrogen sulfide and methanethiol, which may contribute to the generation of off-flavours in yogurt, thereby negatively impacting the desired flavour profile [38]. This shift in ORP can hinder the overall sensory quality of the product due to the impact of these volatile compounds on flavour and aroma [39]. The metabolic activity of these bacteria affects the flavour profile by modulating the balance between the oxidation and reduction reactions that control the generation and conversion of volatile compounds. These metabolic characteristics illustrate the dynamic interplay between redox potential, metabolic pathways, and oxidative stress in influencing the fermentation capabilities of these two lactic acid bacteria in yogurt production.
The proteolysis of S. thermophilus is initiated by the PrtS enzyme, which hydrolyzes caseins into peptides. These peptides promote bacterial growth by providing essential nitrogen sources and stimulating metabolic pathways that initiate fermentation and contribute to milk acidification by promoting lactic acid production, ultimately lowering the pH of the milk [4,10,11]. In this study, there was no significant difference among isolate clusters regarding the time needed to reach a pH of 4.6 and CEP activity; however, the maximum acidification rate was significantly different. Two of the four clusters of S. thermophilus contained faster acidifying isolates with higher proteolytic and CEP activity. Studies have found that the fast acidifying phenotype of S. thermophilus is linked to its PrtS-mediated proteolytic activity, which breaks down caseins into peptides and free amino acids. These products not only support bacterial growth but also accelerate lactic acid production, leading to a rapid decrease in pH. This acidification facilitates milk coagulation by destabilizing casein micelles, which is a step in fermentation important for gelation [10,11,14]. However, rapid acidification during fermentation can weaken the protein–protein bonds between caseins, leading to excessive gel shrinkage and syneresis [40,41]. This results in decreased yogurt quality, with a watery texture and compromised structural integrity. Conversely, slower acidifying strains tend to produce yogurt with higher viscosity, reduced post-acidification, and a flavour profile with acetaldehyde and diacetyl, which many consumers find desirable. As preferences for flavour can vary, the intensity of the yogurt aroma can be modulated by the combination of fast or slow acidifying bacterial strains, with the caveat being that some combinations may lead to excessive sulfur-like aromas that are generally considered undesirable [37,41,42,43].
The activity of aminopeptidases or X-prolyl dipeptidyl peptidases releases peptides or amino acids [44]. Fast acidifying S. thermophilus has previously been shown to be positively correlated with growth rate in the presence of the prtS gene [45,46]. However, in this study, all highly proteolytic S. thermophilus isolates performed as slow acidifiers, whereas L. bulgaricus, with a fast acidification phenotype, showed a wide range of proteolytic activities. Slow acidifying L. bulgaricus strains have been shown to produce yogurt with a thicker texture due to their increased synthesis of exopolysaccharides (EPSs) during fermentation, which thickens and stabilizes the yogurt matrix [47]. This extended fermentation time not only allows for greater EPS production before significant acid accumulation but also contributes to a creamier texture and a more stable protein network [41,48]. Studies on the activity of S. thermophilus strains have shown that strains with high proteolytic activity can exhibit slower acidifying activity because the breakdown of proteins into peptides and amino acids requires energy, which diverts resources from acid production, thus slowing the overall acidification rate [44]. Moreover, Hafeez et al. [43] determined that PrtS- strains can acidify milk at a similar rate to PrtS+ strains, not solely due to the cell envelope protease but through the activity of extracellular peptidases. These enzymes, including aminopeptidases and X-prolyl dipeptidyl peptidases, break down peptides into smaller molecules that the bacteria can utilize, supporting growth and indirectly contributing to acidification through enhanced metabolism [49]. Their findings suggest that the isolates in this study, which showed lower cell-envelope proteinase (CEP) activity due to the absence of certain proteinases, could be compensated by having higher proteolytic activity through the action of peptidases. This higher peptidase activity may explain the efficient release of free amino acids observed in our results, despite reduced CEP activity [44,50,51]. Recent studies have found that the growth of prtS-deficient strains could be attributed to extracellular peptidase activity and two other cell surface proteases, HtrA and SepM, which are responsible for cell surface proteolysis [13,52]. Further studies on the extracellular and intracellular peptidases of these isolates may help evaluate proteolytic activity in combination with the capacity for acidification. Characterization into extracellular peptidases could also help further elucidate the mechanism of the fast acidifying phenotype of Prt- isolates.
Each cluster contained fast and slow isolates, suggesting that the capacity for acidification is partially independent from other phenotypic measures. Erkus et al. [15] studied the carbohydrate utilization phenotype and found phenotypic and genotypic heterogeneity among 22 S. thermophilus isolates from artisanal Yuruk yogurt. They also found that the phenotypic and genotypic profiles could not be differentiated between strains with fast and slow acidifying abilities [15]. Yamauachi et al. [29] found that some strains of L. bulgaricus required CO2 for effective acidification and growth in milk in the presence of nitrogen. Furthermore, ammonia directly provided by the urease activity of S. thermophilus did not affect the growth of L. bulgaricus in milk. Arioli et al. [53] found that acidification by non-growing S. thermophilus cells increased with ammonia and boosted the process of glycolysis, revealing another layer of glycolytic regulation. Further studies on CO2 fixation, urease activity and ammonia production could solve the disparity between the fast and slow acidification phenotypes of each of these species.
S. thermophilus and L. bulgaricus have been shown to engage in complex metabolic cross-feeding and cooperative growth dynamics that involve not just the exchange of growth-promoting metabolites but also the regulation of gene expression and metabolic pathways that optimize their mutual growth [52,53]. Their cooperative relationship enhances the efficiency of fermentation and contributes to the desired sensory properties of yogurt, demonstrating an interaction that extends beyond basic metabolic traits. Compatibility between S. thermophilus and L. bulgaricus may be determined by other phenotypic traits than those used to cluster the isolates in this study. Previous research has indicated that factors such as the production of exopolysaccharides, bacteriocins, hydrogen peroxide and specific metabolic interactions such as the cross-feeding of nutrients, competition for substrates, and the synergistic production of fermentation byproducts during fermentation play significant roles in the compatibility and overall performance of mixed cultures [52,53,54]. This suggests that the compatibility of these strains could be influenced by a network of interactions that are not captured by single or even multi-phenotypic analyses. Moreover, other factors of amino acid catabolism, lipolysis, volatile sulphur compound production, exopolysaccharide synthesis, and specific enzyme activities, all of which contribute to the complexity of flavours and aromas in fermented products, may be more influential in predicting the aroma profiles of the isolates. Therefore, future research should explore a broader range of factors, including genetic and metabolomic profiles, to better understand the determinants of strain compatibility in yogurt starter cultures.
5. Conclusions
This study analyzed ten industrially relevant phenotypic traits of yogurt starter bacteria, focusing on their sensory properties and the overall compatibility between S. thermophilus and L. bulgaricus isolates, established by the absence of direct growth inhibition. The variation in acidification, proteolytic activity, as well as redox potential indicate that a complex interplay of phenotypic diversification can contribute to modulating the performance of starters, either positively or negatively. The isolate compatibility or aroma profiles could not be predicted by the set of 10 industrially relevant traits and appear to be independent of this classification. The multi-phenotype approach should be expanded to metabolic flux mapping to better understand strain behaviour during mixed fermentation. The phenotypic variation observed in the isolates from three distinct starter communities used for yogurt production highlights the necessity of understanding traits such as the acidification rate, proteolytic activity, and redox potential for optimizing texture and flavour. By strategically combining isolates with complementary functional profiles, such as pairing fast acidifying strains with those that enhance creaminess through protein breakdown due to proteolysis, manufacturers can create yogurt products that cater to consumer preferences. This comprehensive approach to phenotypic characterization not only improves product quality but also emphasizes the importance of informed co-culture decisions in the yogurt industry. Future studies should explore the use of machine learning and new combinations of isolates to optimize consumer-preferred aroma characteristics using the volatile profiles of mixed cultures.
Conceptualization, G.L.; Data curation, G.L.; Formal analysis, M.S.; Funding acquisition, G.L.; Investigation, M.S., Z.-H.C. and G.L.; Methodology, M.S., A.T. and Z.-H.C.; Project administration, G.L.; Resources, G.L.; Software, M.S.; Supervision, A.T. and G.L.; Validation, A.T. and G.L.; Writing—original draft, M.S. and G.L.; Writing—review and editing, A.T. and G.L. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
The datasets for this study are available in the Agri-Environmental Research Data Repository of the University of Guelph at
The authors declare no conflicts of interest.
Footnotes
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Figure 1. Average acidification profile of each cluster of (a) S. thermophilus isolates, where each cluster represented as ST1 is [Image omitted. Please see PDF.], ST2 is [Image omitted. Please see PDF.], ST3 is [Image omitted. Please see PDF.], and ST4 is [Image omitted. Please see PDF.], and (b) L. bulgaricus isolates, where each cluster represented as LB1 is [Image omitted. Please see PDF.], LB2 is [Image omitted. Please see PDF.], LB3 is [Image omitted. Please see PDF.], LB4 is [Image omitted. Please see PDF.], and LB5 is [Image omitted. Please see PDF.].
Figure 2. Non-metric multidimensional scaling plot (NMDS) of (a) S. thermophilus monocultures (b) and L. bulgaricus monocultures according to cluster analysis using the Euclidean distance metric and Ward criterion based on 10 variables, including all phenotypic measures (redox potential, acidification activity, proteolytic activity, CEP activity and titratable acidity). Each colour represents a hierarchical cluster at a 25% similarity cut-off. Ellipses represent the 95% confidence interval. Each symbol shape represents an aroma feature after a 16 h fermentation. All samples were tested in triplicate (n = 3).
Cluster analysis of activity parameters for acidification, redox potential, proteolytic activity, and titratable activity of S. thermophilus isolates cultured in homogenized microfiltered milk with a 3.25% fat content. The Euclidean distance metric and Ward criterion were used for the hierarchical clustering procedure based on 10 variables.
Activity Parameter | Cluster of S. thermophilus Isolates | |||
---|---|---|---|---|
ST1 (8) | ST2 (12) | ST3 (12) | ST4 (4) | |
Eh7min (mv) | +38.6 ± 37.7 a,* | −99.2 ± 29.6 b | −71.3 ± 20.5 b | −80.8 ± 38.4 b |
tmin (h) | 10.1 ± 3.76 a | 8.23 ± 1.89 a | 8.53 ± 2.36 a | 7.34 ± 2.22 a |
Δmax | 63.9 ± 40.1 b | 96.7 ± 17.4 a | 31.6 ± 15.7 c | 46.7 ± 8.49 bc |
t* (h) | 9.99 ± 2.47 a | 6.28 ± 2.01 b | 6.90 ± 2.79 b | 5.10 ± 0.70 b |
tph4.6 (h) | 11.4 ± 2.66 a | 12.1 ± 3.74 a | 12.2 ± 1.42 a | 11.5 ± 3.40 a |
Vmax (ph/h) | −0.25 ± 0.04 a | −0.14 ± 0.92 a | −0.19 ± 0.53 ab | −0.20 ± 0.05 ab |
Proteolytic activity | 79.4 ± 38.3 a | 61.7 ± 31.9 a | 76.6 ± 28.8 a | 83.0 ± 35.0 a |
CEP activity | 310 ± 21.4 b | 300 ± 51.8 b | 305 ± 10.3 b | 422 ± 66.3 a |
% lactic acid (1 day) | 0.81 ± 0.27 a | 0.72 ± 20.8 ab | 0.63 ± 24.9 b | 0.67 ± 20.3 ab |
% lactic acid (7 day) | 1.00 ± 0.20 a | 0.90 ± 0.003 a | 0.86 ± 0.005 ab | 0.82 ± 0.011 b |
* Lower-case letters (a, b, c) indicate significant differences between clusters (p < 0.05) using the Dunn–Sidak method for pairwise comparisons between each phenotypic variable. Data represent the mean ± standard error of three replicates. ST1: S1 (n = 3), S2 (n = 2), S3 (n = 3), ST2: S1 (n = 6), S2 (n = 2), S3 (n = 4), ST3: S1 (n = 2), S2 (n = 6), S3 (n = 4), ST4: S1 (n = 1), S2 (n = 2), S3 (n = 1).
Cluster analysis of activity parameters for acidification, redox potential, proteolytic activity, and titratable activity of L. bulgaricus isolates cultured in homogenized microfiltered milk containing 3.25% fat. The Euclidean distance metric and Ward criterion were used for the hierarchical clustering procedure based on 10 variables.
Activity Parameter | Cluster of L. bulgaricus Isolates | ||||
---|---|---|---|---|---|
LB1 (n = 11) | LB2 (n = 9) | LB3 (n = 2) | LB4 (n = 7) | LB5 (n = 7) | |
Eh7min (mv) | −107 ± 21.8 c,* | −85.4 ± 7.73 bc | +90.3 ± 22.9 a | −47.4 ± 7.62 b | −43.2 ± 27.5 b |
tmin (h) | 5.20 ± 1.15 a | 6.32 ± 1.08 a | 7.17 ± 0.77 a | 6.10 ± 2.51 a | 5.77 ± 0.93 a |
Δmax | 36.1 ± 24.4 a | 28.9 ± 12.5 a | 55.5 ± 15.6 ab | 50.0 ± 38.8 b | 50.3 ± 23.8 a |
t* (h) | 4.55 ± 1.56 a | 5.38 ± 3.89 a | 4.27 ± 0.79 a | 3.80 ± 2.35 a | 4.30 ± 0.93 a |
tph4.6 (h) | 7.93 ± 2.20 a | 8.98 ± 3.15 a | 9.37 ± 1.62 a | 9.21 ± 3.01 a | 7.78 ± 1.70 a |
Vmax (ph/h) | −0.27 ± 0.03 a | −0.28 ± 0.35 b | −0.26 ± 2.42 a | −0.32 ± 0.05 a | −0.27 ± 0.40 a |
Proteolytic activity | 51.2 ± 11.9 b | 54.0 ± 18.1 b | 110 ± 1.41 a | 47.1 ± 83.0 b | 109 ± 25.3 a |
CEP activity | 301 ± 40.4 d | 553 ± 51.8 a | 451 ± 10.3 b | 376 ± 66.3 c | 300 ± 44.8 d |
% lactic acid (1 day) | 1.09 ± 0.61 ab | 1.18 ± 0.18 ab | 1.06 ± 0.00 a | 1.19 ± 0.01 ab | 1.31 ± 0.008 b |
% lactic acid (7 day) | 1.23 ± 0.26 ab | 1.23 ± 0.36 ab | 1.12 ± 0.36 a | 1.27 ± 0.11 ab | 1.38 ± 0.21 b |
* Lower-case letters (a, b, c, d) indicate significant differences between clusters (p < 0.05) using the Dunn–Sidak method for pairwise comparisons between each phenotypic variable. Data represent the mean ± standard error of three replicates. LB1: S1 (n = 2), S2 (n = 3), S3 (n = 6), LB2: S1 (n = 2), S2 (n = 4), S3 (n = 3), LB3: S1 (n = 2), S2 (n = 0), S3 (n = 0), LB4: S1 (n = 6), S2 (n = 0), S3 (n = 1), LB5: S1 (n = 0), S2 (n = 4), S3 (n = 3).
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References
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Abstract
The mutualistic relationship between Streptococcus thermophilus (S. thermophilus) and L. delbrueckii subsp. bulgaricus (L. bulgaricus) is responsible for milk coagulation, gel formation, and the flavour of yogurt. Under set-style yogurt processing conditions, the performance of a mixed culture composed of these species depends on key technological parameters such as the capacity for acidification and proteolytic activity. This study aimed to determine the extent of phenotypic diversity by comparing the key traits of acidification and proteolytic activity among isolates found in yogurt starter cultures. Seventy-two isolates from three industrial starter cultures were ranked by either their fast or slow acidification activity (time to reach pH 4.6, 16 h), proteolytic activity, cell envelope proteinase (CEP) activity, redox potential and titratable acidity. The integration of multiple phenotype measures by hierarchical clustering and non-metric dimensional scaling (NMDS) clustered groups of isolates by multifactor similarity. A significant difference (p-value < 0.05) was observed between the clusters regarding redox potential and the proteolytic activity of both S. thermophilus and L. bulgaricus. The integration of multiple phenotypes points to the diversification that may have occurred over repeated culturing of yogurt starter bacteria. The phenotypic diversity may explain the divergence in starter performance and be used to refine the formulation of new starter cultures. Future work will investigate the correlation between the activity of specific enzymes based on the phenotype to explain the separation between the fast and slow acidification of isolates.
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Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer