Streptococcus thermophilus: Metabolic Properties, Functional Features, and Useful Applications

Abstract

Streptococcus thermophilus is a Gram-positive, homofermentative lactic acid bacterium classified within the Firmicutes phylum, recognized for its probiotic properties and significant role in promoting human health. This review consolidates existing understanding of its metabolic pathways, functional metabolites, and diverse applications, highlighting evidence-based insights to enhance scientific integrity. S. thermophilus predominantly ferments lactose through the Embden-Meyerhof-Parnas pathway, resulting in L(+)-lactic acid as the primary end-product, along with secondary metabolites including acetic acid, formic acid, and pyruvate derivatives. Exopolysaccharides (EPS) are composed of repeating units of glucose, galactose, rhamnose, and N-acetylgalactosamine. They display strain-specific molecular weights ranging from 10 to 2000 kDa and contribute to the viscosity of fermented products, while also providing antioxidant and immunomodulatory benefits. Aromatic compounds such as acetaldehyde and phenylacetic acid are products of amino acid catabolism and carbohydrate metabolism, playing a significant role in the sensory characteristics observed in dairy fermentations. Bacteriocins, such as thermophilins (e.g., Thermophilin 13, 110), exhibit extensive antimicrobial efficacy against pathogens including Listeria monocytogenes and Bacillus cereus. Their activity is modulated by quorum-sensing mechanisms that involve the blp gene cluster, and they possess significant stability under heat and pH variations, making them suitable for biopreservation applications. In food applications, S. thermophilus functions as a Generally Recognized as Safe (GRAS) starter culture in the production of yogurt and cheese, working in conjunction with Lactobacillus delbrueckii subsp. bulgaricus to enhance acidification and improve texture. Specific strains have been identified to mitigate lactose intolerance, antibiotic-related diarrhea, and inflammatory bowel diseases through the modulation of gut microbiota, the production of short-chain fatty acids, and the inhibition of Helicobacter pylori. The genome, characterized by a G + C content of approximately 37 mol%, facilitates advancements in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology and heterologous protein expression, with applications extending to non-dairy fermentations and the development of postbiotics. This review emphasizes the adaptability of S. thermophilus, showcasing the variability among strains and the necessity for thorough preclinical and clinical validation to fully utilize its potential in health, sustainable agriculture, and innovation. It also addresses challenges such as susceptibility to bacteriophages and limitations in proteolytic activity.

1. Introduction

Lactic acid bacteria (LAB) play a crucial role in the production of various traditional fermented foods, encompassing dairy items like yogurt, cheese, and kefir, in addition to non-dairy fermented products such as sourdough bread, sausages, sauerkraut, olives, natto, and beverages including beer and wine [1,2,3]. Microorganisms are increasingly acknowledged for their positive contributions to human and animal health, including the reduction in chronic disease prevalence, improvement of nutritional status, modulation of immune responses, promotion of growth, and alleviation of stress [4,5,6,7,8]. The characteristics highlighted emphasize the importance of LAB in various industries, such as food production, medicine, pharmaceuticals, and cosmetics.

LAB produce a range of functional metabolites, such as organic acids, hydrogen peroxide, short-chain fatty acids, bacteriocins, aromatic compounds, bioactive peptides, and exopolysaccharides (EPS) [1,9]. These metabolites impart specific sensory and functional characteristics to fermented foods, including enhanced texture, visual attractiveness, flavor profile, and potential health advantages. Nonetheless, the utilization of LAB as natural food additives is constrained by factors including strain-specific traits and the intended application of the fermented product [10,11].

Streptococcus thermophilus is a notable species within LAB, recognized for its considerable potential in both industrial and health-related applications [12]. The genus Streptococcus includes more than 100 species, among which are pathogenic strains like S. pyogenes and S. pneumoniae. Historically, S. thermophilus was categorized as a subspecies of S. salivarius (S. salivarius subsp. thermophilus) based on its genetic resemblance to S. salivarius and S. vestibularis. In 1991, it was reclassified as a separate species based on findings from DNA-DNA hybridization studies [13]. Throughout millennia, S. thermophilus, in conjunction with species like Lactobacillus delbrueckii and L. lactis, has experienced genetic adaptations that facilitate its survival in milk-based environments [14]. The adaptations involve the inactivation of particular genes associated with glucose metabolism and the reduction or elimination of virulence factors, which differentiates S. thermophilus from pathogenic Streptococcus species.

S. thermophilus is acknowledged as a food-safe microorganism by the European Food Safety Authority (EFSA) and is commonly found in dairy products including yogurt, cheese, and sour cream, in addition to specific fermented plant-based foods [15]. Strains exhibiting enhanced fermentation characteristics are chosen as industrial starter cultures, frequently in conjunction with Lactobacillus species, for the manufacturing of yogurt and cheese [16,17]. With an estimated global consumption of 10²¹ colony-forming units (CFU) annually and a market value of approximately $40 billion [18], S. thermophilus is recognized as the second most economically significant LAB species, following Lactococcus lactis. The versatility and safety profile of S. thermophilus suggest it as a viable candidate for overcoming the limitations of current probiotic LAB. This underscores the necessity to identify and characterize novel strains to satisfy the changing requirements of food production and health applications [19].

Based on the critical discussion in the preceding paragraphs derived from the available scientific published literature, there are specific research gaps that urgently need to be addressed for human welfare and the innovative efforts undertaken by scientists and researchers globally. Therefore, the present review article has been formulated. Consequently, this review aims to thoroughly clarify the taxonomic classification, physiological characteristics, and metabolic adaptability of S. thermophilus, emphasizing its synthesis of bioactive compounds including organic acids, exopolysaccharides, aromatic volatiles, and bacteriocins. This study seeks to systematically assess the functional applications of S. thermophilus in various sectors. It will explore its role as a starter culture in both dairy and non-dairy food fermentations, its probiotic and therapeutic potential in medical applications such as immunomodulation, anti-inflammatory effects, and pathogen inhibition, as well as its growing utility in biotechnology, including genetic engineering for improved metabolite production and postbiotic development. This review integrates contemporary genomic, metabolomic, and clinical findings to elucidate the health benefits, technological merits, and strain-specific differences in S. thermophilus. It also addresses significant challenges, including susceptibility to bacteriophages, survivability in the gastrointestinal tract, and scalability in non-dairy applications, while suggesting future research avenues to enhance its industrial, nutritional, and biomedical applications.

2. Streptococcus thermophilus Classification

S. thermophilus is classified within the phylum Firmicutes, class Bacilli, order Lactobacillales, and family Streptococcaceae. It is classified within the salivarius subgroup of the viridans streptococci, which includes S. salivarius and S. vestibularis [19]. This species is characterized as a nonpathogenic, Gram-positive, non-motile coccus, generally observed as spherical or ovoid cells measuring 0.7–0.9 µm in diameter. Cells are organized in pairs or in extended chains consisting of 10 to 20 cells [13].

S. thermophilus is a homofermentative lactic acid bacterium that primarily produces L(+)-lactic acid via lactose metabolism. This organism is classified as a facultative anaerobe, is negative for catalase, and does not possess cytochrome-mediated electron transport systems that are essential for cellular respiration [13]. Optimal growth is observed at temperatures between 40 and 42 °C, with a viable temperature range extending from 20 to 52 °C; the organism can withstand brief exposure to 60 °C for a duration of 30 min. Growth is suppressed at a temperature of 10 °C and in media with a concentration of 2% NaCl. The bacterium does not hydrolyze arginine, generates ammonia from urea, and shows restricted proteolytic activity [20]. The genomic DNA exhibits a guanine-cytosine (G + C) content of roughly 37 mol% [21]. When cultured on media containing blood, S. thermophilus exhibits either α-hemolytic or non-hemolytic characteristics. The cell wall is composed of peptidoglycans of the Lys-Ala_2–3 type, which play a role in maintaining its structural integrity [21].

S. thermophilus exhibits a preference for lactose, glucose, and fructose as carbon sources, while its metabolism of mannose, maltose, sucrose, melibiose, and galactose is limited [19,22,23,24,25]. Most strains metabolize glucose solely through lactose uptake, resulting in the excretion of galactose into the medium because of suboptimal galactose metabolism (Figure 1). S. thermophilus hydrolyzes casein in milk, where free amino acids are limited, to produce peptides and amino acids necessary for growth [22].

The bacterium has a complex proteolytic system similar to that of other LAB, which includes an extracellular serine protease (PrtS), transport mechanisms for amino acids and peptides, and intracellular peptidases [19,22]. PrtS, belonging to the chymotrypsin family, is attached to the cell wall in specific strains of S. thermophilus and facilitates casein hydrolysis, thereby promoting milk acidification and cell growth [23]. In monoculture within milk, PrtS plays a crucial role in growth. In co-cultures involving protease-positive L. delbrueckii subsp. bulgaricus, S. thermophilus is capable of utilizing peptides that are released through the proteolytic activity of L. bulgaricus, which accounts for the lack of the prtS gene in certain strains [24,25]. The synergistic interaction highlights the ecological adaptability of S. thermophilus in fermented dairy environments.

3. Metabolomic Compounds of Streptococcus thermophilus

3.1. Organic Acids

The acidification rate of milk by S. thermophilus strains in industrial dairy fermentation is a critical parameter that has substantial technological implications for product quality and process efficiency [2,25,26]. The rate is closely associated with the bacterium’s capacity to metabolize different carbohydrates, which has a direct impact on fermentation performance [27]. Research indicates that S. thermophilus reliably utilizes glucose, lactose, and fructose across various strains, highlighting their importance in its metabolic processes. The utilization of various carbohydrates, including galactose, mannose, sucrose, maltose, melibiose, and raffinose, differs considerably among strains, indicating a range of metabolic pathways that influence fermentation results [28].

Lactose, the main carbohydrate found in milk and dairy products, is transported into S. thermophilus cells through galactoside permease, an enzyme associated with the membrane that is encoded by the lacY gene located within the lac operon [29]. Lactose is hydrolyzed intracellularly by β-galactosidase (EC 3.2.1.23, systematic name β-D-galactoside galactohydrolase) into glucose and galactose. Glucose is then metabolized to pyruvate through the Embden–Meyerhof–Parnas pathway, which is followed by its conversion to lactic acid by lactate dehydrogenase [30] (Figure 2). In the majority of strains, galactose and lactic acid are released into the extracellular environment. Certain strains express galactokinase, which catalyzes the phosphorylation of galactose to form galactose-1-phosphate. Depending on the strain, this may be further converted to glucose-1-phosphate or galactose-6-phosphate, facilitating additional lactic acid production [31].

Lactic acid serves as the main fermentation product of S. thermophilus; however, acetic acid is generated as a secondary metabolite, with its concentration influenced by strain-specific traits, culture conditions, and the growth phase [32,33]. The observed variability highlights the intricate nature of S. thermophilus metabolism and its capacity to adjust to environmental influences.

Furthermore, S. thermophilus produces pyruvate formate lyase (EC 2.3.1.54), which facilitates the transformation of pyruvate into formic acid in anaerobic environments [34]. Formic acid plays a crucial role in the growth and metabolism of S. thermophilus and also facilitates the metabolic activity and proliferation of co-cultured bacteria, such as L. delbrueckii subsp. bulgaricus, during yogurt fermentation, thereby enhancing microbial synergy and product characteristics [11].

3.2. Polysaccharides

Numerous strains of S. thermophilus generate EPS, which can be categorized into capsular polysaccharides that are closely linked to the cell surface, and free polysaccharides that are released as loosely associated, viscous substances, commonly referred to as “ropy” or “gely” EPS, into the surrounding environment [35,36]. Although EPS production does not directly promote the growth or survival of S. thermophilus in milk, its in situ synthesis by this species and other LAB plays a significant role in the textural characteristics of fermented dairy products, providing desirable viscosity and a smooth, creamy consistency [37,38]. Recent studies indicate that EPS-producing S. thermophilus strains contribute to the enhancement of functional and sensory characteristics in fermented dairy products, including Mexican Panela cheese [39] and yogurt [40]. Nonetheless, these strains continue to exhibit susceptibility to bacteriophage infections, which may interfere with EPS production and impact product quality [41].

The molecular weight of S. thermophilus EPS exhibits significant variation, spanning from 10 kDa to 2000 kDa, which indicates the structural diversity inherent in these biopolymers [42]. The chemical composition and structure of EPS are specific to each strain and generally comprise repeating units of monosaccharides, such as D-galactose, D-glucose, L-rhamnose, and N-acetylgalactosamine. The physicochemical properties of EPS, including viscosity and gelation, are influenced by these components, which are essential for their functionality in food and biotechnological applications (

Table 1. Types of exopolysaccharides produced of S. thermophilus.

Strain Types	Growth Conditions	Sub Units	Function	References
S. thermophilus CGMCC 7.179	The bacterium was cultured in LM17 broth for a duration of 24 h at a temperature of 42 °C.	Mannose, glucuronic acid, galacturonic acid, glucose and N-acetylglucosamine	Antioxidant activity	[43]
S. thermophilus S6-13	The strain was cultured in MRS broth at a temperature of 37 °C for a duration of 16 h.	Glucose, galactose, and N-acetylglucosamine	Higher viscosity of yogurt	[44]
S. thermophilus ZJUIDS-2-01	The strain was cultured in both MRS broth and M17 at 37 °C for 12 h.	Glucose, galactose, N-acetyl-D-galactosamine, and rhamnose	Antioxidant activity and antibacterial properties	[45]
S. thermophilus NQ12	The strain underwent anaerobic incubation for a duration of 24 h at a temperature of 40 °C in an M17 medium supplemented with 0.1% lactose.	Galactosamine, galactose, glucose and mannose	Viscosity factor	[46]
S. thermophilus 90301	2% (v/v) of bacteria were cultured in MRS medium with an inoculation rate maintained for 24 h at a temperature of 37 °C.	Mannose, rhamnose, glucosamine, glucose, and galactose	Potential prebiotic	[47]
S. thermophilus CH9	The strain was incubated at 40 °C for a duration of 24 h on a modified skimmed milk-based medium.	Fucose, ribose, rhamnose, arabinose, xylose, sorbose, glucose, and galactose	Antitumor activity	[48]
S. thermophilus ASCC 1275	Inoculate 1% of the strain into M17 broth that supplemented with 1% lactose, and incubate at 37 °C for a duration of 18 h.	Glucose, galactose, and mannose	-	[49]
S. thermophilus MN-BM-A01	The strain was cultured in skimmed milk at a temperature of 37 °C for a duration of 24 h.	Rhamnose, glucose, galactose, and mannose	Decrease in disease activity index and reduction in colonic epithelial cell injury	[50]
S. thermophilus CRL1190	The strain was cultured in a reconstituted skim milk medium.	Galactose and glucose	Immunoregulatory factor	[51]

). The variability in EPS composition that depends on the strain highlights the intricate biosynthetic pathways and biochemical interactions involved in their production. This complexity renders them useful for specific applications in the food industry, especially in improving texture and stability, as well as in biotechnological fields.

Hyaluronic acid (HA) is a high-molecular-weight, linear heteropolysaccharide that is a significant extracellular polysaccharide produced by specific strains of S. thermophilus. In contrast to other extracellular polysaccharides, HA is characterized by the absence of sulfate groups and is produced through a unique biosynthetic pathway that does not involve Golgi-associated enzymes [52]. Mohammed and Naimah [53] showed that a local S. thermophilus isolate can produce HA when cultured in M17 medium at 42 °C for 48 h, indicating its potential as a natural antioxidant. The distinct characteristics of S. thermophilus EPS, such as viscosity, molecular weight variation, and diverse monosaccharide composition, facilitate their application across multiple domains, including food (e.g., texture enhancement in dairy products), pharmaceuticals (e.g., drug delivery systems), cosmetics (e.g., moisturizing agents), and medical fields (e.g., tissue engineering).

3.3. Aromatic Compounds

S. thermophilus is essential in the formation of the distinctive flavor profile of fermented dairy products by synthesizing a variety of aromatic compounds (Figure 3). The generation of these metabolites primarily results from the interconnected pathways of carbohydrate and amino acid metabolism, with the equilibrium between the two influenced by the enzymatic capabilities of the strain and the composition of the growth medium [54,55]. Acetaldehyde is considered a significant volatile metabolite, contributing to the characteristic fresh, green, and nutty aromas associated with yogurt. The synthesis in S. thermophilus occurs through multiple metabolic pathways. These include the direct decarboxylation of pyruvate by pyruvate decarboxylase, the conversion of pyruvate into acetyl-CoA followed by its reduction to acetaldehyde through the combined actions of pyruvate dehydrogenase and aldehyde dehydrogenase, and the catabolism of threonine by serine hydroxymethyltransferase (SHMT, EC 2.1.2.1), which breaks down threonine into acetaldehyde and glycine. The levels of fermentable carbohydrates, including lactose and sucrose, in the growth medium significantly affect the efficiency of acetaldehyde biosynthesis [56,57].

S. thermophilus plays a role in flavor development through amino acid catabolism, utilizing amino acids primarily obtained from the proteolytic breakdown of milk proteins as substrates for the formation of volatile compounds [25]. The first step in this process generally involves a transamination reaction, in which an amino acid transfers its amino group to an α-keto acid, like α-ketoglutarate, resulting in the formation of a new α-keto acid that acts as an intermediate for subsequent catabolic pathways. A notable metabolic characteristic of S. thermophilus is its activity of glutamate dehydrogenase (GDH, EC 1.4.1.3), which facilitates the endogenous production of α-ketoglutarate from glutamate [58]. The metabolic advantage enables the bacterium to effectively degrade amino acids without the need for externally supplied α-keto acids, a feature not uniformly seen in other LAB. The breakdown of specific amino acids results in the production of various significant volatile compounds, including phenylacetic acid and phenylethylamine derived from phenylalanine, along with other derivatives from tyrosine and tryptophan. However, the range of compounds generated by S. thermophilus is typically more limited compared to that seen in some lactobacilli [15].

Additionally, S. thermophilus contains the shikimate pathway, facilitating the de novo biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. This pathway plays a crucial role in protein synthesis and cellular metabolism; however, its direct impact on the aroma profile of dairy products is comparatively limited when contrasted with the catabolism of preformed amino acids in nutrient-rich environments like milk fermentation. Nonetheless, the pathway is significant in providing essential precursors when these amino acids are in short supply [59,60]. The metabolic processes of S. thermophilus demonstrate its essential function in transforming carbohydrates and amino acids into volatile aromatic compounds, which contribute to the unique sensory characteristics of fermented dairy products like yogurt and cheese.

3.4. Bacteriocins

Various strains of S. thermophilus generate bacteriocins, known as “thermophilins,” which are antimicrobial peptides synthesized by ribosomes and encoded by distinct genetic loci. The peptides demonstrate a range of antimicrobial activities, from narrow to broad, affecting various Gram-positive bacteria and, in certain instances, Gram-negative bacteria (Table 2). Significant targets comprise organisms responsible for food spoilage and foodborne pathogens, including Listeria monocytogenes, Salmonella enterica serovar Typhimurium, Escherichia coli, and Bacillus cereus, among others [3,61,62]. The antimicrobial efficacy of thermophilins differs considerably among S. thermophilus strains and is affected by environmental factors such as growth conditions, nutrient availability, and culture pH [3,61,62].

Thermophilins exhibit notable physicochemical stability, with several showing resilience over a wide pH spectrum (e.g., pH 2–10) and the ability to withstand elevated temperatures, including those encountered during pasteurization (e.g., 100 °C for 30 min) [61,63,64]. The thermal and pH stability renders them especially appropriate for applications in food preservation, where processing conditions may be rigorous. Certain thermophilins demonstrate heat lability, underscoring the structural and functional diversity present within this class of bacteriocins [64].

The biosynthesis of thermophilins is often controlled by quorum-sensing mechanisms, which are mediated by signaling molecules like the bacteriocin-like peptide (BlpC). These systems allow S. thermophilus to adjust bacteriocin production based on cell density, thereby optimizing antimicrobial activity in particular ecological contexts [65]. Genomic analyses have identified significant bacteriocin-encoding genes, including those for thermophilin 13 and thermophilin 110, frequently situated within the “blp” (bacteriocin-like peptide) gene cluster, a conserved locus in S. thermophilus genomes [66,67].

Thermophilins, due to their natural origin and strong antimicrobial properties, are a promising category of biopreservatives that can improve food safety and quality. Their potential extends beyond food applications, with emerging research investigating their therapeutic utility in addressing microbial infections and influencing microbial communities in health-related contexts [66]. Additional examination of their structure-function relationships, variability specific to strains, and regulatory mechanisms will be essential for enhancing their applications in industrial and clinical settings.

Some specific instances of S. thermophilus producing bacteriocins with strong antibacterial characteristics against food-borne infections and antibiotic-resistant varieties are well-documented. Thermophilin 13, derived from strain SFi13, demonstrates inhibitory effects against Clostridium botulinum, Lt. monocytogenes, and B. cereus, indicating its potential application as a biopreservative in dairy products [1,67]. Thermophilin 110, synthesized by strain B59671, exhibits extensive efficacy against Lt. monocytogenes and S. mutans, an opportunistic pathogen found in the oral cavity [66,68]. Thermophilin 1277, derived from strain SBT1277, exhibits a specific affinity for LAB and spoilage organisms, including C. butylicum and B. cereus. Its thermal stability renders it an effective agent for food preservation [69]. Furthermore, a bacteriocin derived from strain 81 demonstrates a broad inhibitory range against multiple foodborne pathogens, such as Listeria and Bacillus species [70]. Bacteriocins present a viable alternative to conventional antibiotics, effectively tackling the issue of multidrug-resistant pathogens in both food safety and health-related applications.

Both early and recent preclinical and clinical studies underscore their effectiveness, safety, and potential for both commercial and therapeutic uses. Research has shown that bacteriocins produced by S. thermophilus, including thermophilin and streptococcin, display extensive activity against foodborne pathogens such as Lt. monocytogenes, E. coli, and Staphylococcus aureus, encompassing multidrug-resistant (MDR) strains [66,71,72]. In vitro assays demonstrate that these bacteriocins interfere with bacterial cell membranes, creating pores that result in cell lysis. This mechanism is considered less prone to inducing resistance in comparison to conventional antibiotics [73]. In vivo investigations, predominantly utilizing murine models, have validated their effectiveness. For example, thermophilin decreased the colonization of Lt. monocytogenes in the gastrointestinal tract of mice, with no significant toxicity noted at therapeutic doses [74]. The results indicate that S. thermophilus bacteriocins may function as potent antimicrobial agents against multidrug-resistant pathogens.

Safety evaluations are essential for the process of clinical translation [75]. Preclinical investigations suggest that S. thermophilus bacteriocins exhibit low toxicity to mammalian cells, which is linked to their preferential targeting of bacterial membranes rather than those of eukaryotic cells [76,77]. In vivo toxicity assessments conducted in mice and other models, such as guinea pigs, indicated no detrimental effects on essential organs or immune responses at concentrations that are effective against pathogens [76,78]. Furthermore, these bacteriocins demonstrate low immunogenicity and cause minimal disruption to gut microbiota, thereby establishing them as safer alternatives to antibiotics, which frequently lead to dysbiosis [79,80].

The main obstacle in the application of bacteriocins, as highlighted in formulation studies, is their vulnerability to proteolytic degradation, which restricts their stability in vivo [1,77,81]. Recent formulation investigations have examined encapsulation methodologies, including liposomes and nanoparticles, to improve stability and bioavailability [62,82,83,84,85,86]. Encapsulation of thermophilin within chitosan nanoparticles has been shown to enhance its resistance to gastric enzymes, thereby prolonging its activity under simulated gastrointestinal conditions [87,88]. These advancements are essential for the formulation of oral or topical applications intended for clinical use [81].

The bacteriocins produced by S. thermophilus exhibit considerable potential in commercial and therapeutic contexts, particularly in food preservation and clinical applications [62,89]. Their recognized application in dairy products, such as yogurt, highlights their commercial potential as biopreservatives, with nisin serving as a prominent example [1,62,89,90]. Their therapeutic potential in targeting multidrug-resistant pathogens, such as Klebsiella pneumoniae and methicillin-resistant St. aureus (MRSA), responds to the critical demand for innovative antimicrobials amid the escalating threat of antimicrobial resistance (AMR), which is anticipated to result in 10 million fatalities each year by 2050 [1,62,90,91,92]. Nonetheless, obstacles persist, such as the expenses associated with large-scale production and the regulatory challenges encountered in obtaining clinical approval [62,90]. Current investigations in bioengineering and synthetic biology focus on improving the yield and spectrum of bacteriocins, which may help to address these challenges [90].

Table 2. Characteristics and applications of bacteriocins produced by S. thermophilus.

Bacteriocin Name	Type/Class	Molecular Weight (Da)/Amino Acids	Key Characteristics	Activity Spectrum (Examples)	References
Thermophilin 13	Class IIb (Two-peptide bacteriocin)	5776 (62 aa, ThmA); 3910 (43 aa, ThmB)	Requires two peptides (ThmA and ThmB) for activity. Genes characterized	Lt. monocytogenes, Bacillus spp., S. thermophilus	[67,70]
Thermophilin 110	Unspecified (likely Class I or II)	-	Heat-stable, broad-spectrum. Production can be influenced by growth media.	Pediococcus acidilactici, other spoilage and food-borne pathogenic bacteria	[65,93]
Bacteriocin from S. thermophilus 81	Unspecified	~32 amino acids	Heat-labile but pH-stable (3–10). Activity not affected by 6 months storage at 4 °C. Inactivated by detergents and proteolytic enzymes.	Bacillus spp., Lt. monocytogenes, Sl. typhimurium, E. coli, Yersinia pseudotuberculosis, Y. enterocolitica, L. delbrueckii subsp. bulgaricus.	[70]
BlpU (from strain B59671)	Class II (encoded within blp gene cluster)	~5–6 kDa (heat-stable peptide)	Broad-spectrum. Production regulated by quorum sensing (BlpC induction peptide). Inactivated by protease treatment.	Enterococcus faecalis, E. faecium, L. helveticus, S. mutans, S. salivarius, B. cereus, S. pyogenes	[94]
Bacteriocin from S. thermophilus LMD-9 (Thermophilin 9 related)	Class II (Multiple peptides, blp locus dependent)	-	Inhibitory spectrum dependent on multiple peptides and the activity of BlpGSt. Quorum-sensing regulated.		[95]
Bacteriocin from S. thermophilus ACA-DC 0001	Unspecified	-	Heat-unstable (activity lost at 60 °C for 1 h).	Not specified, but likely similar to other St. thermophilus bacteriocins.	[96]
Bacteriocin from S. thermophilus CHCC 3534	Unspecified	14.4 to 18.4 kDa (partially purified)	Heat-stable, pH-resistant, inactivated by proteolytic enzymes, resistant to α-amylase and lipase.	Broad antimicrobial spectrum against St. aureus Sl. typhimurium.	[97]
Bacteriocin from S. thermophilus ST109	Class II (blp gene cluster encoded)	~5–6 kDa (heat-stable)	Production regulated by BlpC (quorum sensing). Inactivated by protease and α-amylase.	Lactobacilli, enterococci, S. pyogenes.	[70]

Table 2. Characteristics and applications of bacteriocins produced by S. thermophilus.

Bacteriocin Name	Type/Class	Molecular Weight (Da)/Amino Acids	Key Characteristics	Activity Spectrum (Examples)	References
Thermophilin 13	Class IIb (Two-peptide bacteriocin)	5776 (62 aa, ThmA); 3910 (43 aa, ThmB)	Requires two peptides (ThmA and ThmB) for activity. Genes characterized	Lt. monocytogenes, Bacillus spp., S. thermophilus	[67,70]
Thermophilin 110	Unspecified (likely Class I or II)	-	Heat-stable, broad-spectrum. Production can be influenced by growth media.	Pediococcus acidilactici, other spoilage and food-borne pathogenic bacteria	[65,93]
Bacteriocin from S. thermophilus 81	Unspecified	~32 amino acids	Heat-labile but pH-stable (3–10). Activity not affected by 6 months storage at 4 °C. Inactivated by detergents and proteolytic enzymes.	Bacillus spp., Lt. monocytogenes, Sl. typhimurium, E. coli, Yersinia pseudotuberculosis, Y. enterocolitica, L. delbrueckii subsp. bulgaricus.	[70]
BlpU (from strain B59671)	Class II (encoded within blp gene cluster)	~5–6 kDa (heat-stable peptide)	Broad-spectrum. Production regulated by quorum sensing (BlpC induction peptide). Inactivated by protease treatment.	Enterococcus faecalis, E. faecium, L. helveticus, S. mutans, S. salivarius, B. cereus, S. pyogenes	[94]
Bacteriocin from S. thermophilus LMD-9 (Thermophilin 9 related)	Class II (Multiple peptides, blp locus dependent)	-	Inhibitory spectrum dependent on multiple peptides and the activity of BlpGSt. Quorum-sensing regulated.		[95]
Bacteriocin from S. thermophilus ACA-DC 0001	Unspecified	-	Heat-unstable (activity lost at 60 °C for 1 h).	Not specified, but likely similar to other St. thermophilus bacteriocins.	[96]
Bacteriocin from S. thermophilus CHCC 3534	Unspecified	14.4 to 18.4 kDa (partially purified)	Heat-stable, pH-resistant, inactivated by proteolytic enzymes, resistant to α-amylase and lipase.	Broad antimicrobial spectrum against St. aureus Sl. typhimurium.	[97]
Bacteriocin from S. thermophilus ST109	Class II (blp gene cluster encoded)	~5–6 kDa (heat-stable)	Production regulated by BlpC (quorum sensing). Inactivated by protease and α-amylase.	Lactobacilli, enterococci, S. pyogenes.	[70]

4. Application of Streptococcus thermophilus

4.1. In Food

S. thermophilus serves as a fundamental organism in the dairy fermentation sector, possessing a documented history of safe application in food manufacturing, which has led to its designation as Generally Recognized as Safe (GRAS) by regulatory bodies including the United States Food and Drug Administration (FDA) [98]. The primary application of this organism is in the production of yogurt, where it is commonly utilized in a synergistic co-culture with L. delbrueckii subsp. bulgaricus. This symbiotic relationship improves the growth and metabolic efficiency of both strains, thereby optimizing fermentation outcomes [99]. S. thermophilus efficiently converts lactose, the main sugar found in milk, into lactic acid. This biochemical process facilitates the coagulation of milk proteins, leading to the distinctive thick texture and tangy flavor associated with high-quality yogurt [100,101].

S. thermophilus is an essential starter culture utilized in the production of a range of cheeses, such as Mozzarella [102], Swiss-type cheeses [103], processed cheeses [104], and reduced-fat cheeses [105]. The function of these products includes acidification and the synthesis of EPS, which play a significant role in achieving the intended texture, mouthfeel, and flavor enhancement. The bacterium demonstrates acidification capabilities in various fermented dairy products, including sour cream and cultured milk beverages, contributing to improved sensory attributes and product stability.

Recent studies indicate the adaptability of S. thermophilus in non-dairy contexts, especially in the fermentation processes involving plant-based materials such as fruit and vegetable juices, including barley juice. This process enhances flavor profiles and increases the content of bioactive compounds, including phenolics and flavonoids, which in turn boosts antioxidant activity [74]. The characteristics of S. thermophilus indicate its potential as a candidate for the development of functional foods that possess improved nutritional profiles. Recent developments in genetic engineering and genomic analysis have broadened the capabilities of S. thermophilus as a microbial host for the production of heterologous proteins and various biotechnological applications [106]. The genome is well-characterized, and the metabolic properties are robust, rendering it suitable for the engineering of novel functional food ingredients, such as probiotics with specific health benefits. S. thermophilus remains a crucial microorganism in both conventional and novel food fermentation processes, with increasing acknowledgment of its probiotic potential and its role in human health.

4.2. In Medicine

The probiotic potential of S. thermophilus is still being examined due to its inconsistent resilience in the gastrointestinal tract; however, certain strains are acknowledged as probiotics based on comprehensive in vitro and in vivo research. These strains play a role in maintaining gut microbiome balance by generating bioactive compounds, which include folate, bacteriocins exhibiting antimicrobial properties, and antioxidant enzymes like superoxide dismutase. Furthermore, S. thermophilus strains demonstrate anti-inflammatory properties that may influence immune responses and reduce gastrointestinal inflammation (Figure 4).

S. thermophilus promotes the synthesis of short-chain fatty acids (SCFAs), including butyrate and acetate, which are essential for preserving gut barrier integrity and modulating immune function. Clinical evidence indicates its effectiveness in reducing antibiotic-associated diarrhea (AAD) and symptoms of inflammatory bowel disease, such as ulcerative colitis and Crohn’s disease, by restoring microbial balance and decreasing intestinal inflammation [74,106]. The bacterium produces lactase, which improves lactose digestion and provides significant benefits for individuals with lactose intolerance by enhancing their ability to tolerate dairy products [107].

The therapeutic applications of S. thermophilus include a reduction in the severity and duration of acute diarrhea and antibiotic-associated diarrhea, as evidenced by randomized controlled trials [108,109]. S. thermophilus mitigates microbial dysbiosis caused by antibiotics or infections through competitive inhibition of pathogens and the restoration of eubiosis [110]. In irritable bowel syndrome, S. thermophilus, frequently used in conjunction with other probiotics such as Lactobacillus spp., has demonstrated the potential to reduce symptoms including bloating, flatulence, and irregular bowel movements, although results can differ based on strain and individual patient characteristics.

Preclinical studies, especially those involving animal models, indicate that S. thermophilus may offer protection against chronic gastritis through the modulation of immune responses and the preservation of the gastric mucosal barrier [111,112,113]. Specific strains of S. thermophilus, when utilized alongside standard therapies, have demonstrated enhanced eradication rates of Helicobacter pylori in clinical environments, which may lead to a decrease in the occurrence of gastritis and peptic ulcers [114]. The anti-inflammatory properties of S. thermophilus, noted in both live and postbiotic forms (such as secreted metabolites), play a role in its potential therapeutic applications for managing inflammatory conditions, including sepsis, through the modulation of gut microbiota composition and function [115,116].

Additionally, S. thermophilus contributes to immune system function by fostering a balanced gut microbiota, which is essential considering that around 70% of immune cells are situated in the gut-associated lymphoid tissue. The antimicrobial properties may inhibit pathogens in the gastrointestinal and urogenital tracts, thereby enhancing host defense mechanisms [117]. S. thermophilus is classified as GRAS by regulatory authorities; however, its clinical efficacy varies by strain, and current research is focused on identifying the most effective therapeutic applications. Formulations that combine different probiotic strains frequently improve functional benefits, highlighting the significance of strain selection and combinatorial strategies in clinical applications.

4.3. In Biotechnology

S. thermophilus is an important microorganism in biotechnology, primarily utilized in the dairy industry as a key starter culture for the production of yogurt and cheese. The GRAS status, granted by regulatory authorities, supports its extensive application and encourages investigation into new biotechnological uses. The metabolic versatility of the organism, along with advancements in genomic and genetic engineering tools, has greatly increased its applications beyond conventional dairy fermentation.

Genomic studies of S. thermophilus have clarified its metabolic pathways, especially those related to carbohydrate utilization, proteolysis, and metabolite production, which are essential for industrial applications [118]. Essential metabolic pathways, including lactose and galactose metabolism, play a significant role in its swift acidification abilities, whereas proteolytic systems facilitate the production of peptides that improve flavor and texture in fermented products. Recent studies have concentrated on refining these pathways to enhance technological characteristics, including the production of EPS for increased viscosity, faster acidification rates, and improved flavor profiles.

The advancement of advanced genetic tools has transformed the engineering of S. thermophilus. Plasmid-based expression systems, along with inducible promoters, transcriptional terminators, and genome-editing technologies such as CRISPR-Cas systems, facilitate accurate modifications to the genome [119]. The advancements facilitate the enhancement of traits pertinent to industrial applications, including elevated EPS yields for improved texture in dairy products, as well as the introduction of new functionalities, such as the biosynthesis of bioactive compounds. Additionally, S. thermophilus is being recognized as a potential host for the production of heterologous proteins, presenting a feasible alternative to other LAB such as Lactobacillus species, owing to its strong metabolic capabilities and GRAS designation [120].

S. thermophilus is being investigated for its potential in fermenting non-dairy substrates, including barley juice, with the aim of enhancing nutritional profiles, improving sensory attributes, and increasing antioxidant activity via the production of bioactive metabolites [121]. Furthermore, the postbiotics, which consist of non-viable microbial cells or their components, have attracted interest due to their possible anti-inflammatory and immunomodulatory effects, leading to potential applications in functional foods and nutraceuticals [122].

The adaptability of S. thermophilus, supported by its GRAS designation and varied metabolic functions, establishes it as a significant resource in the field of biotechnology. Current developments in genetic engineering and synthetic biology are expected to enhance its application in creating novel pharmaceutical uses, reinforcing its significance in both industrial and therapeutic settings, food products, health-enhancing components, and possible.

5. Recent Trends of Streptococcus thermophilus Applications

S. thermophilus is a lactic acid bacterium commonly employed in the manufacturing of fermented dairy products, including yogurt and cheese, owing to its strong metabolic functions and probiotic characteristics. Recent research has broadened its applications beyond conventional dairy systems, emphasizing its incorporation into non-dairy functional foods, independent probiotic formulations, and innovative biotechnological applications, demonstrating its versatility and adaptability.

The increasing adoption of plant-based diets has led to a rise in the use of S. thermophilus for the fermentation of non-dairy substrates, including plant-based yogurts, fruit juices, and cereal-based beverages. These applications are designed to improve nutritional profiles, sensory characteristics, and functional properties. Fermentation of barley juice using S. thermophilus has been demonstrated to enhance flavor profiles and increase antioxidant activity, which aids in the creation of health-promoting beverages [121]. In a similar manner, its application in legume- and nut-based matrices improves texture and bioaccessibility of nutrients, responding to consumer demand for functional, plant-based alternatives.

Recent findings highlight the ability of S. thermophilus to influence host health through its probiotic properties. Certain strains have shown the capacity to assimilate cholesterol in vitro, indicating a possible function in promoting cardiovascular health through the reduction in serum cholesterol levels [106]. S. thermophilus plays a role in maintaining gut homeostasis by enhancing the intestinal barrier, regulating immune responses, and favorably affecting the composition of gut microbiota. The characteristics of S. thermophilus suggest its potential as a candidate for standalone probiotic supplements, with current research investigating strain-specific mechanisms and clinical efficacy [106,122].

A notable trend in the research of S. thermophilus is the investigation of its postbiotic potential, which includes bioactive compounds like EPS, bacteriocins, and short-chain fatty acids generated during fermentation. These compounds maintain bioactivity post-bacterial inactivation, providing health advantages including anti-inflammatory and antimicrobial properties. This has generated interest in postbiotics derived from S. thermophilus as functional components for food fortification and pharmaceutical applications, with potential roles in gut health, immune support, and disease prevention [74,122].

The extraction of indigenous S. thermophilus strains from particular geographical areas or traditional fermented products has become increasingly relevant for its contribution to the preservation of cultural heritage and the improvement of product authenticity. The specific strains play a significant role in defining the distinct sensory characteristics of artisanal cheeses, especially those recognized with Protected Designations of Origin (PDO). Researchers are utilizing the genetic diversity of indigenous strains to preserve the organoleptic and microbiological integrity of traditional fermented foods, all while adhering to contemporary safety and quality standards [123].

Recent developments in genetic engineering and synthetic biology have established S. thermophilus as a viable platform for various biotechnological applications. The genetic malleability allows for the creation of customized strains that exhibit enhanced functional characteristics, including increased probiotic efficacy and optimized production of bioactive metabolites. These advancements are facilitating the development of new applications in personalized nutrition, targeted therapeutics, and sustainable food production systems.

S. thermophilus continues to be a fundamental component of the dairy sector, with recent developments emphasizing its growing involvement in non-dairy fermentation, probiotic supplementation, postbiotic creation, and biotechnological advancements. The observed trends highlight the adaptability of the bacterium and its capacity to meet changing consumer demands for functional, sustainable, and health-enhancing food products, along with its developing uses in pharmaceutical and biotechnological fields.

6. Conclusions

S. thermophilus, a significant species within the lactic acid bacteria category, demonstrates notable metabolic flexibility and functional characteristics that support its extensive application in food manufacturing, health enhancement, and biotechnological advancements. This review consolidates evidence indicating that S. thermophilus generates a variety of bioactive metabolites, such as organic acids (including lactic, acetic, and formic acids), EPS, aromatic compounds (like acetaldehyde and phenylacetic acid), and bacteriocins (such as thermophilins). These metabolites collectively improve the sensory, nutritional, and preservative attributes of fermented products, while also providing antimicrobial, antioxidant, anti-inflammatory, and immunomodulatory benefits. The metabolic pathways of S. thermophilus, including the EMP route for glucose catabolism and the Leloir and Tagatose pathways for galactose utilization, facilitate effective carbohydrate fermentation and acid production, which are essential for dairy acidification and symbiotic relationships with species such as L. delbrueckii subsp. bulgaricus. EPS variants, defined by their strain-specific compositions (such as glucose, galactose, and rhamnose), enhance the texture and viscosity of fermented foods, while also exhibiting bioactivities that include prebiotic potential and antitumor effects. Aromatic compounds resulting from the catabolism of amino acids and carbohydrates play a significant role in defining flavor profiles. Additionally, bacteriocins such as thermophilin 13 and 110 demonstrate broad-spectrum antimicrobial activity against foodborne pathogens, including Lt. monocytogenes and B. cereus, as well as multidrug-resistant strains, with their regulation occurring through quorum-sensing mechanisms. The applications of S. thermophilus are diverse and span multiple fields. In the food industry, it is recognized as a GRAS-designated starter culture for yogurt, cheese, and innovative non-dairy fermentations, such as plant-based juices, where it contributes to improved nutritional bioavailability and enhanced antioxidant properties. In the medical realm, S. thermophilus functions as a probiotic that alleviates conditions like antibiotic-associated diarrhea, irritable bowel syndrome, and infections caused by H. pylori, primarily through the modulation of gut microbiota and the maintenance of immune homeostasis. Furthermore, in biotechnology, it serves as a genetically amenable host for the expression of heterologous proteins and the production of postbiotics, utilizing CRISPR-Cas technologies for the optimization of desired traits. Recent trends highlight the growth of S. thermophilus within plant-based matrices, independent probiotics, and postbiotics, propelled by consumer interest in sustainable, functional food options. Nonetheless, obstacles remain, such as variability in metabolite yield that is specific to different strains, vulnerability to bacteriophages, and the requirement for improved proteolytic systems in non-dairy settings. Future investigations ought to focus on genome-wide association studies to clarify regulatory mechanisms, bioengineering aimed at enhancing resilience and bioactivity, and clinical trials to confirm therapeutic efficacy, thus optimizing its impact on human health, food security, and industrial sustainability.