Introduction

Probiotics are widely used in functional dairy products because of their health benefits and consumer acceptance (Hasnain et al., 2024; Jang et al., 2024). However, their viability is often compromised during processing, storage, and gastrointestinal (GI) transit, limiting product efficacy (D’Amico et al., 2025). Protein conjugation has emerged as a promising strategy to enhance probiotic stability and functionality, primarily through the Maillard reactions and emerging technology–assisted or chemical approaches. Such conjugates protect probiotics from environmental stress, improve intestinal adhesion, and enhance dairy product quality (Vivek et al., 2023), positioning protein–probiotic interactions as a key innovation in functional food design.

The escalating demand for functional dairy products has stimulated growing interest in integrating probiotics with protein–based technologies to enhance nutritional and health benefits. Emerging processing approaches, such as ultrasound, ohmic heating, and high–pressure treatment, have been investigated for their effects on probiotic viability and metabolic performance (Gavahian et al., 2021). In this context, dairy–derived proteins have become key functional ingredients in food, clinical, and pharmaceutical formulations owing to their exceptional reconstitution, emulsifying, and foaming properties, which surpass those of lipids and carbohydrates (Wu et al., 2021). Beyond their physicochemical roles, protein hydrolysates have demonstrated prebiotic potential by supporting probiotic proliferation (Guo et al., 2022), while hydrogel–based protein–polysaccharide complexes have been developed for targeted probiotic delivery in therapeutic applications (Zheng et al., 2023). Complementary investigations conducted by Wu et al. (2024) have provided molecular insights into plant protein–probiotic interactions and underscored the structural and compositional constraints of plant–based matrices. Despite substantial progress in probiotic delivery and functional dairy research, few studies have systematically elucidated how the chemical nature and formation parameters of protein conjugates influence the survival, metabolic activity, adhesion, and host interactions of probiotics within dairy systems. Current reviews often address these aspects in isolation; for instance, Xiang et al. (2021) examined the effects of the Maillard reaction on milk proteins without linking these effects to probiotic functionality, underscoring the need for integrative perspectives on protein conjugate–probiotic interactions.

Recent research has demonstrated that under unfavorable conditions, Maillard–type conjugates formed from milk proteins, such as whey or casein, combined with carbohydrates, such as dextran or glucose, can enhance the viability of probiotics. For instance, Guo et al. (2022) demonstrated that whey protein (WP)–dextran encapsulated conjugates enhanced the heat and acid resistance of Lactiplantibacillus plantarum 21805 in yogurt formulations. Similarly, Wu et al. (2021a) demonstrated that glycosylation of whey protein via controlled dry heating of skim milk powder significantly enhanced protein thermal stability under clean–label conditions, enabling recombined evaporated milk emulsions to withstand heating at 120°C for 30 min without loss of stability. A’yun et al. (2020) reported a great improvement in the heat stability of whey protein isolate (WPI)–lactose conjugates, which could successfully stabilize oil–in–water (O/W) emulsions against heating (80°C for 20 min). In addition, the Maillard conjugates of whey protein hydrolysates (WPH) and linear dextrins (LD; degree of polymerization [DP] 23.78–64.96) were prepared by Pan et al. (2020) by dry heating, with glycation degree increasing with LD content. Glycation altered WPH secondary structure, reduced α–helix content, and enhanced antioxidant activity. Additionally, conjugation of casein–derived hydrophobic peptides with Acacia seyal polysaccharides markedly enhanced the stability of O/W emulsions (Hou et al., 2017). Thus, the Maillard–type protein–carbohydrate conjugates, such as those formed from whey, casein, or serum albumin with sugars such as dextran or glucose, hold strong potential to enhance probiotic stability and functionality in dairy products, even under heat, acid, and GI stress.

This review summarizes recent advances in protein conjugation strategies, evaluates their effects on probiotic functionality in dairy products, and highlights their potential for developing next–generation dairy products with improved health benefits and nutritional value. The review discusses key health impacts, including modulation of the gut microbiota, enhancement of the immune system, increased bioavailability of bioactive peptides, and antioxidant properties. Finally, current technological and regulatory challenges are considered along with future perspectives for the commercial application and continued development of protein–conjugation techniques in probiotic dairy products.

Overview of Probiotics and Protein Conjugation

Probiotics in dairy foods

Probiotics, defined as live microorganisms that confer health benefits when consumed in adequate amounts, remain a key focus of modern health food research (Zavišić et al., 2023; Gao et al., 2021; Hill et al., 2014). Probiotic food sources have their historical roots in fermented dairy products, including cheese and yogurt, which were traditionally produced through spontaneous fermentation by lactic acid bacteria (LAB) (Kaur et al., 2022), and are valued for their taste, shelf life, and promotion of intestinal health (Jang et al., 2024). Élie Metchnikoff, “father of innate immunity,” linked regular consumption of fermented dairy products to improved immunity, longevity, and many biochemical processes (Lee et al., 2025). Over time, scientific knowledge about probiotics has evolved to the point that the concept has expanded beyond microorganisms derived from food to include bacteria isolated from the human gastrointestinal tract (GIT), particularly those capable of promoting health and maintaining intestinal homeostasis (Zommiti et al., 2020).

Today, probiotics are widely available in various forms, including functional foods, nutritional supplements, and pharmaceutical preparations. Owing to their advantageous physicochemical characteristics, including buffering capacity, nutritional richness, and compatibility with microbial growth and survival, dairy products remain the most widely used and effective carriers of probiotics (Latif et al., 2023). This also reflects the worldwide acceptance of dairy–based probiotics as promoting overall health, lifespan, and disease prevention (Kaur et al., 2022).

Applications derived from dairy products benefit from a wide range of probiotic species. Among these, the most commonly employed genera include Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Enterococcus, Leuconostoc, and Propionibacterium (Vivek et al., 2023). A broad spectrum of strains, such as Lactobacillus acidophilus, Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, L. plantarum, Limosilactobacillus reuteri, B. bifidum, Bifidobacterium longum, and Bifidobacterium lactis, are regularly included in yogurt, fermented milk, cheese, and milk powders (Vivek et al., 2023). Importantly, there is no consensus on the lowest effective dose of probiotics; however, a reasonable daily intake of approximately 10⁹ colony–forming units (CFU), taken consistently, is usually considered sufficient to confer health benefits (Latif et al., 2023).

In addition to probiotics, nondigestible food ingredients, known as prebiotics, are sometimes added to enhance the effectiveness of most fermented dairy products. Probiotic species, including Bifidobacterium and Lactobacillus, break down prebiotics, such as inulin, fructooligosaccharides (FOS), and galactooligosaccharides (GOS), thereby improving gut microbiota composition and mucosal immunity (Wang et al., 2020). Consequently, in synbiotic formulations, probiotics and prebiotics exhibit synergistic effects that promote colonization, enhance microbial survival, and yield more physiological outcomes in the host (Parhi et al., 2024). A comprehensive overview of probiotic microorganisms used in various dairy products and their applications are presented in Table 1.

Dairy products are among the most established and successful vehicles for probiotics in the food industry because of their inherent nutrient content, favorable physicochemical properties, and consumer acceptance (Jang et al., 2024). In particular, LAB play a vital role in ensuring microbiological safety, shelf life, and sensory quality of fermented dairy products by synthesizing organic acids and antimicrobial peptides and competing with pathogens through competitive inhibition (Ibrahim et al., 2021). Milk provides an ideal medium for delivering viable probiotic cultures because of its buffering capacity, nutritional density, and protein structure, which protect probiotics during GI transit (Gao et al., 2022). Among fermented dairy products, yogurt is the most commonly consumed and investigated vehicle for probiotic bacteria. Moreover, the sector has recently expanded to include probiotic–enriched fermented dairy products, such as pasteurized milk, ice cream, and infant formula, which use microencapsulation or post–fermentation addition techniques to ensure probiotic viability during storage (Sbehat et al., 2022).

Various studies have demonstrated that probiotic dairy products can regulate gut bacteria, improve lactose digestion, enhance the immune system, and alleviate manifestations of GI diseases (Mudgil et al. 2024a). The combinations of probiotics in both fermented and nonfermented dairy matrices reflect growing innovation in the design of functional dairy foods. These products are developed for their nutritional value and targeted health benefits, including relief from lactose intolerance, immune modulation, and gut microbiota balance. However, maintaining probiotic viability throughout processing, distribution, and storage remains a key challenge, particularly for nonfermented products, such as ice cream and infant formula. Therefore, to fully harness the health benefits of probiotic dairy products, especially nonfermented types, it is essential to enhance the stability of probiotics across processing and storage.

Protein–probiotic conjugation mechanisms

Protein conjugates interact with probiotics at the molecular level to enhance gastrointestinal stability, adhesion, and colonization via multiple biochemical and structural mechanisms. These strategies enable targeted delivery (Liu et al., 2025), enhance stress tolerance (Zhang et al., 2023a), and improve therapeutic efficacy in the gut environment (Peled et al., 2024; Yin et al., 2025). Table 2 presents key protein–probiotic conjugation mechanisms.

The initial improvements in probiotic survival, adhesion, and localization are primarily driven by direct protein–probiotic interactions, whereas the major therapeutic outcomes, such as production of short–chain fatty acids (SCFA), immune modulation, and disease relief, are largely mediated by secondary microbiome–driven effects (Liu et al., 2025; Peled et al., 2024). In practice, full efficacy results from synergistic coupling of both mechanisms, rather than from either pathway alone. Furthermore, despite the potentiality of these mechanisms in the protein–probiotic interactions, they approach challenges related to strain specificity, environmental variability within the gut, and long–term microbiome interactions (Song et al., 2022; Zhang et al., 2023a), thus paving the way for further study, such as validation through in vivo to show feasibility for clinical translation.

Protein conjugation techniques used in dairy systems

Protein conjugates refer to covalently bonded complexes formed between proteins and other biomolecules, such as carbohydrates, polyphenols, or similar compounds, through various chemical or enzymatic processes, enhancing the functional properties of the resulting complexes. Structurally and functionally, protein–sugar conjugates can shield probiotics from environmental stresses, increase their adherence to the intestinal epithelium, and even improve the sensory quality of the final dairy product (Vivek et al., 2023; Zhang et al., 2023b). Conjugation can alter the physicochemical and biological characteristics of milk proteins. The growing demand for functional dairy products has therefore driven substantial advances in protein modification technologies. Among the various techniques available, studies have identified three main approaches: the Maillard–type conjugation, enzymatic cross–linking, and physical activation, which currently hold the greatest potential. The methods and parameters used in recent studies on protein–carbohydrate conjugates in dairy products are presented in Table 3.

The Maillard, enzymatic, and chemical conjugation each provides distinct mechanistic avenues for improving probiotic functionality in dairy systems, although their benefits and limitations diverge. The Maillard conjugation, driven by the covalent attachment of reducing sugars to lysine residues, reinforces protein matrix stability while simultaneously enhancing the protective barrier against acid and bile stress (Gao et al., 2021; Zhang et al., 2023b). By contrast, enzymatic conjugation, most notably via microbial transglutaminase (MTGase), exhibits greater substrate specificity, producing ε–(γ–glutamyl)–lysine cross–links that not only stabilize probiotic encapsulation but also enhance mucosal adhesion and promote the controlled release of bioactive peptides during digestion (Milczek, 2018). Chemical conjugation expands versatility by enabling the covalent grafting of proteins with polyphenols or polysaccharides to form complexes that provide strong shielding against environmental stressors; however, concerns over off–flavors, altered digestibility, and regulatory approval limitations restrict their translational potential (Zhang et al., 2023a).

Polysaccharide–protein complexes (PPCs) offer significant advantages over single–protein carriers in dairy systems, serving as biocompatible alternatives to synthetic emulsifiers (Sun et al., 2022). They enhance colloidal stability during processing and storage, improve the encapsulation and bioaccessibility of bioactive compounds, and positively modify textural and sensory attributes, collectively leading to greater consumer acceptance of dairy products (Liu et al., 2025). Despite these functional benefits, the industrial application of PPCs is constrained by scalability and reproducibility challenges. Protein–polysaccharide interactions are highly sensitive to processing conditions, such as pH and ionic strength, often resulting in variability in complex formation and performance (Goh et al., 2020). Furthermore, maintaining consistent quality during large–scale production and meeting clean–label and regulatory requirements remain critical hurdles that must be addressed to enable broader adoption of PPCs in commercial dairy systems (Liu et al., 2025).

Maillard–protein conjugation

The Maillard reaction is a nonenzymatic glycation process in which reducing sugars react with protein amino groups to form Schiff bases, ultimately leading to stable ketoamine linkages (Figure 1) (Liu et al., 2020). In dairy systems, this reaction is typically performed under dry or semi–dry conditions at moderate temperatures (50–80°C) and approximately 70% relative humidity (RH) for 24–72 h (da Silva et al., 2019). The reaction alters protein structures by exposing hydrophilic groups and increasing surface activity, thereby enhancing their functional and protective roles. It has been widely applied to milk proteins, including WPI (Xu et al., 2019), casein (Yuan et al., 2024), and milk protein concentrate (Wu et al., 2021b), with saccharides, such as inulin, dextran, Arabic gum, and xylooligosaccharides (XOS)/GOS. While this processing can improve the nutritional quality and quality of foods, it requires precise control. Excessive heating may cause undesirable browning, nutritional losses of essential amino acids, and the formation of advanced glycation end products (AGEs), which are increasingly concerning for health–conscious consumers. For the same reason, Bi et al. (2023) recommended exposure at 70°C for 15 s as optimal conditions to preserve both quality and nutritional value in a study on the effect of pasteurization on the Maillard reaction in lactose–free milk.

Figure 1. Schematic illustration of protein–carbohydrate conjugation pathways. Physical pretreatments followed by dry or wet heating promote Maillard conjugation, enhancing the functional and bioactive properties of the resulting protein–carbohydrate complexes.

The Maillard conjugation substantially improves the functional properties of milk proteins, including solubility, heat stability, emulsifying ability, and antioxidant activity (Xu et al., 2019). These enhancements directly translate into improved probiotic survival during GI stress and thermal processing, making this approach particularly valuable for yogurt and fermented milk products. In such matrices, Maillard–modified proteins not only protect probiotic cells but also enhance texture and provide prebiotic benefits, supporting synbiotic formulations. Consistently, higher counts of Lactobacillus and Bifidobacterium have been reported in conjugate–fortified dairy, compared with nonconjugated carriers (Chaiwong et al., 2025; Liao et al., 2021).

Extending this strategy to plant–based proteins presents challenges because of structural heterogeneity, reduced solubility, and lower lysine availability, all of which can hinder conjugation efficiency. For example, pea protein–polydextrose conjugates produced by wet heating have been shown to improve solubility, emulsifying, and foaming properties, with the protein–to–polydextrose ratio, temperature, and time as key factors (Khan et al., 2024). Similarly, soy protein isolate–oligosaccharide Maillard conjugates enhanced the antioxidant activity of the carrier and improved Lactobacillus casei survival under simulated digestion, maintaining 7.4 log CFU/mL, compared to 1.6 log CFU/mL in free cells (Zhong et al., 2021). Moreover, Nghiep (2024) found that optimized energy–nitrogen combinations enhance nutrient value and protein efficiency, affecting feed intake and nutrient digestibility in Saanen crossbred goats, suggesting that the conjugation effect of protein–polysaccharides is of great importance in dairy systems. Despite such progress, plant–based Maillard reaction products (MRPs) still fall short of the probiotic protection typically achieved with dairy carriers, underscoring the need for further optimization of conditions and formulations.

Importantly, the Maillard conjugation utilizes food–safe materials and provides multifaceted functionality without relying on synthetic additives. It improves solubility, emulsification, antioxidant activity, and probiotic stability (Chaiwong et al., 2025). Nonetheless, uncontrolled reactions may induce browning, off–flavors, and the formation of AGEs, which pose nutritional and health concerns. When carefully managed, however, the Maillard–protein conjugation remains a powerful strategy for probiotic delivery, particularly in dairy applications (Dong et al., 2023; Kathuria et al., 2023). Moreover, recent work has highlighted the potential of two–stage complexation–covalent conjugation followed by complex coacervation as a promising route to engineer more stable protein–carbohydrate systems (Zhang et al., 2023b).

Enzymatic protein–conjugation

Enzymatic protein conjugation involves specific biocatalysts that form covalent bonds between proteins or between proteins and other biopolymers, thereby tailoring their functional and structural properties. Among these, MTGase is the most widely used enzymatic conjugation method in dairy systems. Other enzymes, such as laccases, tyrosinases, and peroxidases, catalyze protein–polyphenol or protein–protein cross–linking, potentially conferring functional benefits in plant–based systems due to their broader substrate ranges (Gouseti et al., 2023). MTGase catalyzes the formation of ε–(γ–glutamyl) lysine bonds via acyl transfer between glutamine and lysine residues (Milczek, 2018), thereby generating larger, more stable protein networks. Reactions usually occur under mild conditions (30–50°C, near–neutral pH) and are completed within 1–4 h (da Silva et al., 2019), making the process compatible with probiotic viability. In dairy matrices, MTGase is effective at low doses, is safe, and enhances the texture of yogurt and cheese by strengthening casein gels, thereby improving viscosity and gel strength, and enabling physical entrapment of probiotic cells (da Silva et al., 2019; Milczek, 2018). Furthermore, Halavach (2024) demonstrated that enzymatic hydrolysis of whey and colostrum proteins using alcalase and protozyme (5%, 50–60°C) generated low–molecular–weight peptides (<10 kDa) with high immunoglobulin hydrolysis (82.4%). These hydrolysates are potentially hypoallergenic and suitable for functional foods and nutraceutical applications. Compared to chemical cross–linking, enzymatic conjugation offers advantages in precision, safety, and the avoidance of unwanted by–products, such as AGEs (Kathuria et al., 2023).

Despite the advantages of enzymatic conjugation in dairy systems, the cost of enzymes, batch–to–batch variability in activity, and inhibition by food components (e.g., metal ions, phenolics, or extreme pH levels) may limit scalability (Kathuria et al., 2023). Additionally, although MTGase efficiently cross–links caseins owing to their flexible, open structures, plant proteins (e.g., soy, pea, and oats) pose greater challenges. Their globular conformation and reduced lysine/glutamine accessibility reduce conjugation efficiency (Chen et al., 2020), often necessitating pretreatment (e.g., denaturation, hydrolysis, or high–pressure processing) to expose reactive sites. This distinction highlights a crucial difference: applying the same approach to plant–based matrices often compromises sensory quality by producing excessive gel rigidity and off–flavors, or necessitates higher enzyme inputs, thereby increasing regulatory concerns (Sim et al., 2021). However, such effects may be more tolerable in plant–based alternatives (Santoso et al., 2024; Yi et al., 2024). Therefore, a critical balance must be achieved between functionality and sensory acceptance. Furthermore, future research should critically compare enzyme systems across both domains, tailoring approaches to optimize reactivity while preserving desirable sensory attributes.

Chemicals–protein conjugation

Chemical conjugation, which has limited applications in commercial dairy products due to regulatory constraints, employs external cross–linking agents to form covalent bonds between protein molecules or to graft functional groups, such as polyphenols, oligosaccharides, or polysaccharides (Figure 2), onto proteins (Zanjani et al., 2018). These reactions target nucleophilic functional groups on protein surfaces, including amines, carboxyls, thiols, and phenolic residues. Among these, common food–compatible cross–linkers include genipin (from Gardenia jasminoides), EDC (1–ethyl–3–(3–dimethylaminopropyl) carbodiimide), and oxidized polysaccharides with reactive aldehyde groups (Wei and Huang, 2019; Zanjani et al., 2018).

Figure 2. Schematic illustration of dextran–protein conjugate formation via periodate oxidation and reductive amination. Dextran is first oxidized by sodium periodate to produce aldehyde groups, which subsequently react with the amino groups of proteins to form Schiff bases. The imine linkages are then reduced by sodium borohydride (NaBH₄) to yield stable covalent dextran–protein conjugates.

Chemical conjugation approaches in dairy applications are primarily limited to food–grade, safe cross–linking agents. For example, genipin–cross–linked protein matrices have been utilized to encapsulate probiotic strains, such as L. reuteri, thereby enhancing their viability during spray drying and exposure to simulated GI fluids (Laurujisawat et al., 2025). Additionally, chemical grafting of proteins to dietary fibers (e.g., pectin or alginate derivatives) or polyphenolic compounds (e.g., tannic acid and catechins) can create protein–polyphenol or protein–fiber complexes with protective, antioxidant, and antimicrobial properties within the constraints of food safety regulations (Wei and Huang, 2019).

Protein conjugation via chemical methods offers high versatility and stability, enabling the design of multifunctional delivery systems resistant to acid, heat, and mechanical stress. However, potential limitations include off–flavors, discoloration (especially with genipin), and reduced digestibility of excessively cross–linked proteins. Additionally, the regulatory status of some cross–linkers may pose challenges, as only specific agents, such as genipin, are approved for food use, and any unreacted chemicals must be removed or proven unsafe (Laurujisawat et al., 2025; Wei and Huang, 2019; Zanjani et al., 2018). Thus, chemical–protein conjugation offers strong functional benefits but is limited by regulatory restrictions, sensory changes, and the need for safe, food–grade cross–linkers.

Mechanism of interactions of protein conjugates in the dairy system

The description of the Maillard, enzymatic, and chemical conjugation techniques provides an overview of their synthesis; however, a mechanistic understanding is essential to explain how these conjugates interact with probiotic cell surfaces. At the molecular level, electrostatic attraction, hydrophobic interactions, and hydrogen bonding are primarily responsible for binding between conjugated biopolymers and bacterial membranes (Liu et al., 2023; Sun et al., 2023). The cationic amino acid residues of proteins in the conjugates can interact with the negatively charged teichoic acids, peptidoglycan, and exopolysaccharides on the probiotic cell walls, whereas the hydrophobic regions anchor to the lipid components of the membrane (Sun et al., 2022). In Maillard– and enzymatically derived conjugates, reactive carbonyl and amino groups can form weak covalent or hydrogen–bonding interactions with surface proteins or polysaccharides, thereby enhancing adhesion stability (Liu et al., 2023). Additionally, surface–layer (S–layer) proteins, lipoteichoic acids, and extracellular polysaccharides of Lactobacillus and Bifidobacterium species act as specific binding sites that mediate these interactions (Bönisch et al., 2018; Du et al., 2023). The conjugate–cell–surface associations reinforce membrane integrity, reduce permeability to environmental stressors, and enhance probiotic resilience during processing and GI transit (Sun et al., 2023).

This study confirms that the effectiveness of conjugation varies among probiotic strains because of differences in surface proteins and cell wall composition, making the response largely strain–dependent, as highlighted in Table 3. Furthermore, there is no universal ratio; most studies report effective conjugation between 1:1 and 1:3 (w/w), but the optimal proportion depends on the strain and the conjugation method used. The conjugation approaches in this review provide insight into a continuum between functional efficacy and practical applicability, suggesting that future innovation may dwell in hybrid systems that combine the biological precision of enzymatic strategies with the structural resilience of both Maillard and chemical pathways, as indicated by several studies in Table 3.

Role of Protein Conjugates on Probiotic Functionality of Dairy Products

Role of conjugation on the survival of probiotics in dairy products

Probiotics often experience viability loss under various processing conditions, including thermal treatments, spray drying, freeze drying, and prolonged storage (Manyatsi et al., 2024). However, protein conjugation has proven highly effective in addressing these challenges. Several studies have demonstrated that conjugating proteins, such as WPI, sodium caseinate, soy protein isolates, and plant–derived proteins such as peas and rice, with carbohydrates or polyphenolic compounds via various encapsulation processes enhances the proportion of probiotic survival (Xu et al., 2019). Pumpkin carotenoids complexed with casein and soy proteins showed improved stability and functionality after spray drying (Werasakulchai et al., 2025). Protein–carotenoid powders are suitable for use in food. This is consistent with protein conjugation strategies in dairy foods to enhance bioactive delivery.

For example, Liao et al. (2021) prepared the Maillard reaction conjugates of WPI and XOS to encapsulate L. rhamnosus by spray drying. Results showed that WPI–XOS conjugates improved the survival of L. rhamnosus during spray drying with enhanced storage stability (at 4°C) for up to 8 weeks. Similarly, Song et al. (2024) investigated WPI–soybean hull polysaccharide conjugates that encapsulated L. casei by freeze–drying at –80°C. The result showed that probiotics encapsulated with conjugates can survive during processing and storage. This results in higher viability during freeze–drying (87.35% vs. 24.95% for unencapsulated) and better thermal resistance during pasteurization at 60°C and 80°C. Furthermore, ultrasound–assisted multilayer Pickering emulsions prepared from WPI conjugated with (–)–epigallocatechin–3–gallate (EGCG) protected L. plantarum well during pasteurization at 63°C for 30 min. The probiotic survival proportion after pasteurization for the unconjugated sample was 48.5%, compared to 74.25% for the WPI–EGCG conjugated multilayer emulsion. This indicates that the conjugated system improved thermal protection by approximately 25.75% (He et al., 2023).

Additionally, Mudgil et al. (2024b) investigated the stability of L. reuteri DSM 17938 encapsulated within pea and rice protein conjugates with inulin. The results showed that conjugating plant proteins with prebiotic inulin and rice protein achieved the highest percentage of efficiency (96.99%), compared to pea protein conjugates (92.87%). Protein conjugates form dense protective layers around probiotics, shielding them from environmental stresses, including heat, moisture, oxygen, and acidic conditions (Li et al., 2025). Specifically, the Maillard reaction enhances barrier properties by forming stable covalent bonds between proteins and carbohydrates, resulting in robust microcapsules that prevent probiotic degradation during processing and storage (Gao et al., 2021; Liao et al., 2021; Minj and Anand, 2022).

Protection of probiotics against gastrointestinal stressors

During digestion, probiotics encounter several stressors: in the stomach, the environment is highly acidic (pH 1.5–3.0) and contains digestive enzymes, such as pepsin; in the small intestine, exposure to bile salts and pancreatin at near–neutral pH (6.5–7.5) further threatens cell viability; and in the colon, anaerobic conditions and microbial competition present additional survival hurdles (Li et al., 2025; Minj and Anand, 2022; Werasakulchai et al., 2025). To improve probiotic viability, recent research has focused on the use of protein–carbohydrate Maillard conjugates formed by covalent bonding between proteins (e.g., WPI, soy protein isolate, and caseinate) and carbohydrates (e.g., oligosaccharides and polysaccharides) as effective protective encapsulating agents (Du et al., 2023; Liao et al., 2021; Mudgil et al., 2024b). Similarly, Liao et al. (2021) reported enhanced resistance of L. rhamnosus encapsulated in WPI–XOS Maillard conjugates under simulated gastric conditions. When exposed to simulated gastric fluid (pH 2.0, for 2 h at 37°C), free cells exhibited a drastic reduction in viability, dropping to below 4.0 log CFU/g, while encapsulated cells maintained over 7.5 log CFU/g, indicating strong acid resistance and potential for delivery to the intestines.

Moreover, probiotics encapsulated within WPH–maltodextrin conjugates demonstrated improved survival under simulated gastric acid conditions (pH 2.5 for 2 h at 37°C). L. acidophilus encapsulated maintained a viability of 7.4 log CFU/g, compared with a sharp decline to 3.2 log CFU/g in the control. Furthermore, upon exposure to bile salts (0.3% for 3 h at 37°C), the encapsulated cells remained above 7.0 log CFU/g, demonstrating the conjugate’s effectiveness in maintaining probiotic viability across GI conditions (Minj and Anand, 2022). Furthermore, the hydrophilic–hydrophobic balance and viscoelastic gel structure of protein conjugates delay digestive penetration and modulate controlled release, enabling colon–targeted delivery (Gao et al., 2021; Minj and Anand, 2022). The challenge in moving forward exists in optimizing conjugate formulations for large–scale food applications while ensuring safety by avoiding the overproduction of AGEs.

Protein conjugates enhance probiotic survival under GI stress by creating a multi–layered protective matrix that reduces direct exposure of cells to gastric acid, bile salts, and digestive enzymes (Li et al., 2025; Werasakulchai et al., 2025). Mechanistically, conjugated proteins reduce proton permeability and bind bile salts, thereby mitigating membrane disruption in probiotic cells. Studies in simulated GI models demonstrate that whey protein–oligosaccharide conjugates increase survival proportions of Lactobacillus rhamnosus and Bifidobacterium longum, compared to unconjugated carriers (da Silva et al., 2019; Liao et al., 2021; Rolim et al., 2021). Preliminary human intervention studies further suggest that probiotic delivery with whey protein conjugates results in higher recovery of viable Lactobacillus in the small intestine, supporting improved colonization and persistence (Rolim et al., 2021). These findings suggest that protein conjugation strategies not only serve as physical shields but also modulate the digestive microenvironment, thereby creating translational opportunities for next–generation probiotic formulations in functional dairy systems.

Role of Protein Conjugates on the Quality of Dairy Products

Functional quality of dairy products with protein–carbohydrate conjugation

Researchers have demonstrated that conjugated proteins with carbohydrates, formed through the Maillard reaction, can seriously impact the technological and functional properties of the final conjugates, particularly in areas such as solubility, heat stability, emulsification, foaming properties, gelation, and texture (Deng et al., 2025; Urango et al., 2024; Zhang et al., 2023b). Primarily, conjugates made through the Maillard–type glycation or enzymatic cross–linking exhibit amphiphilic characteristics that enable them to serve as protective matrices and functional stabilizers in dairy–based systems. The key function of protein–carbohydrate conjugates in probiotic dairy systems is their ability to act as encapsulating agents. Protein conjugates can form protective matrices around probiotics through microencapsulation processes (Loyeau et al., 2021; Werasakulchai et al., 2025).

Additionally, several studies have demonstrated diverse applications of protein conjugates in dairy products. Mudgil et al. (2025) showed that camel milk protein complexed with polyphenols from dates’ seeds achieved substantially improved emulsification stability and nearly three–fold increase in foaming capacity compared to nonconjugated protein powders. Chaiwong et al. (2025) reported that whey protein–GOS conjugates (1:2 ratio, heated for 6 h at 90°C) exhibited superior solubility and emulsion stability compared to whey protein alone, demonstrating better technological properties than other conjugate ratios tested. Additionally, Akalan et al. (2024) added 1% protein conjugate prepared from brown rice protein and microcrystalline cellulose, or 1% brown rice protein, to set yogurt. The protein conjugate and brown rice protein considerably improved dry matter, water–holding capacity, firmness, consistency, viscosity index, and cohesiveness while decreasing syneresis in yogurt samples compared to plain yogurt. The application of protein conjugates also extends to plant–based dairy alternatives. Soy protein isolate–maltodextrin conjugates can be effectively used as substitutes for conventional proteins in various applications, including enhancing foaming stability and improving solubility (Choi et al., 2025). Similarly, microalgal proteins have emerged as promising ingredients for dairy alternatives, often outperforming terrestrial plant proteins because of their balanced amino acid profile and superior techno–functional properties (Samarathunga et al., 2025). In another investigation on ice cream incorporating WPI and rare sugars (D–allose and D–psicose) for glycation, it was reported that ice cream with WPI glycated with rare sugars had a higher overrun than ice cream with untreated WPI (Puangmanee et al., 2008).

In addition to protecting probiotics, protein conjugates markedly influence the flavor and physicochemical properties of dairy products. Key quality characteristics of yogurt, cheese, and milk–based beverages include enhanced emulsification capacity, gel–forming ability, and water–holding capacity, which define texture, viscosity, and mouthfeel (Mudgil et al., 2025). For instance, conjugates generated by the Maillard reaction between WPI and inulin have been shown in yogurt systems to reduce whey separation and increase product creaminess (Żbikowska et al., 2020). For dried milk, a conjugated WPH with maltodextrin was used as a carrier to protect B. animalis spp. lactis ATCC 27536 and L. acidophilus ATCC 4356 (Minj and Anand, 2022). The protein conjugate provided better probiotic protection during spray drying, resulting in a final probiotic count of 8.98 log CFU/g, compared to an initial count of 10.58 log CFU/mL before drying.

Gut microbiome analysis can be more effectively integrated into protein–probiotic conjugate design by using microbial composition and functional outputs to guide the selection of compatible prebiotics and probiotic strains for targeted outcomes. For instance, nondigestible dextrin, α–cyclodextrin, and dextran selectively enhance acetate and propionate production in human fecal fermentation models, highlighting how microbiome–driven SCFA profiles can inform conjugate design aimed at gut and metabolic health (Sasaki et al., 2018). Clinical evidence further demonstrates the value of microbiome–informed selection, as Lactobacillus–, Bifidobacterium–, and multi–strain formulations, such as a multi–strain formulation comprising four Lactobacillus strains, three Bifidobacterium strains, and one Streptococcus salivarius subsp. Thermophilus strain modulates inflammatory pathways, enhances mucosal barrier function, and reduces systemic markers of inflammation in inflammatory bowel disease and ulcerative colitis (Ganji–Arjenaki and Rafieian-Kopaei, 2018; Groeger et al., 2013). Together, these findings support a targeted design strategy in which microbiome profiling and host biomarkers directly inform the development of protein–probiotic conjugates with predictable anti–inflammatory and gut–protective effects.

Sensory attributes of protein conjugate containing dairy products

Gharibzahedi and Altintas (2024) further reported the sensory properties of set yogurt added with a protein conjugate, showing a slight difference with an optimum condition of 4.65% (w/w) transglutaminase–cross–linked protein isolate and 10.1 log CFU/mL L. acidophilus LA–5^® producing yogurt with maximum lightness, firmness, apparent viscosity, and probiotic survivability, combined with minimum syneresis. Moreover, the yogurt with transglutaminase–cross–linked protein isolate exhibited less sensory deterioration than other yogurt samples during refrigerated storage over 2 weeks.

Nevertheless, the issue of product color after adding the protein conjugate was reported by researchers who supplemented the conjugate in ice cream and cream products. Seo and Yoo (2022b) used milk protein isolate–κ–carrageenan conjugates as part of the whipping cream ingredients and noted that excessively high levels of the conjugates affected the color of the whipping creams, making them appear more yellow A similar drawback was discovered if imitation Mozzarella cheese was supplemented with conjugates of cricket protein and FOS (Chailangka et al., 2023). The findings revealed that imitation Mozzarella cheese with 30% or higher conjugates received lower sensory scores for its appearance, color, odor, texture, flavor, and overall acceptability.

Protein sources in protein–probiotic conjugation

Protein conjugates from both milk and plant sources play important roles in stabilizing probiotics, enhancing their adhesion, and modulating host immunity. However, they function through distinct mechanisms and with varying effectiveness. Milk proteins, such as whey and casein, are particularly effective for microencapsulation, protecting probiotics during processing and GI transit, and their surface properties enhance adhesion to the intestinal mucosa (Abd El–Salam and El–Shibiny, 2015). They also influence gut immunity by promoting beneficial microbiota and SCFA production, thus reducing inflammation (Peled et al., 2024). In contrast, plant proteins primarily interact with exopolysaccharides in fermented products to maintain probiotic structural integrity and support functional effects, including antioxidative and anti–inflammatory activities. However, their encapsulation and immune–modulatory capacities are less well characterized (Zang et al., 2025). These findings suggest that milk–derived proteins offer robust and predictable benefits for probiotic stabilization and immune modulation. In contrast, plant proteins provide complementary advantages in specific applications, such as fermented foods, making the choice of protein system dependent on health goals, product type, and dietary considerations.

Role of Protein Conjugates in Delivering Nutritional and Health Advantages of Probiotics in Dairy Products

Probiotics can take the form of live cultures, inactivated cultures, postbiotics, and prebiotics, and are regarded as integral to functional dairy products. Owing to the presence of probiotics in dairy products, these products functionally improve overall digestive performance by influencing the intestinal microbiota, particularly when combined with protein conjugates. It has been found to confer various health benefits, including alleviating lactose intolerance, modulating the immune system, and controlling cholesterol levels (Mokhtari et al., 2019). Furthermore, it is asserted that probiotics, when combined or encapsulated in protein–conjugated products within a dairy product matrix, could enhance their benefits through the stabilization and delivery of probiotics, thereby increasing their bioavailability and modulating immune responses and gut flora (El Jeni et al., 2024).

Recent studies also suggest that protein–carbohydrate conjugates may enhance the biofunctional activity of probiotic systems. After digestion, these conjugates release bioactive peptides that exhibit antioxidant, antibacterial, and antihypertensive properties (Minj and Anand, 2022). Additionally, oxygen–sensitive bacteria (A. muciniphila) have been encapsulated using emerging delivery methods, including those employing milk exosomes coupled to bioactive proteins. A recent in vitro study in mice has shown that such systems can enhance mucosal immunity and metabolic balance compared with unencapsulated systems (Hao et al., 2025). Protein conjugates also aid in regulating immune responses, thereby enhancing probiotics’ ability to support immune function (Hao et al., 2025). Moreover, they enhance the bioavailability of probiotics and bioactive peptides, thereby maximizing their health benefits (Hadjimbei et al., 2022; Li et al., 2023), as illustrated in Figure 3. Most evidence supporting the benefits of protein–probiotic conjugates remains largely confined to in vitro studies and animal models.

Figure 3. Overview of the functional roles of protein conjugates in probiotic dairy systems, highlighting their contribution to probiotic bioavailability, nutrient absorption, and host health protection. Source: Hadjimbei et al. (2022); Hao et al. (2025); Li et al. (2023); Lim et al. (2024); Rashidi et al. (2021).

Evidence studies on the nutritional and health roles of protein conjugates in probiotic–based dairy products

Recent studies have explored the potential of protein conjugates to enhance the nutritional and health–promoting effects of probiotics in food systems. For instance, in a study conducted by Massounga Bora et al. (2021), WPI and (−)–epigallocatechin–3–gallate conjugates were evaluated as carriers for L. acidophilus, demonstrating enhanced in vitro antioxidant (78%) and antidiabetic (52%) activities as well as improved probiotic survival ability and surface hydrophobicity. Similarly, Yu et al. (2021) examined the effects of casein and chicken–protein diets on recovery from dextran sulfate sodium (DSS)–induced colitis in mice. While DSS caused no physiological differences between the diet groups, the casein diet increased the beneficial gut bacteria. These findings collectively highlight the role of protein–based conjugates and dietary proteins in improving probiotic delivery, physiological functioning, and GI health.

Evidence suggests that protein conjugates enhance the survival, adhesion, and colonization of probiotics by forming protective matrices and promoting the targeted release of bioactive peptides and prebiotic–like sugars (Li et al., 2023; Mudgil et al., 2024a). Mechanistically, MTGase–mediated cross–linking strengthens hydrophobic and electrostatic interactions with intestinal epithelium, while the Maillard–derived reductones and melanoidins provide antioxidant protection to both probiotics and the host. Rashidi et al. (2021) reported that human clinical trials have demonstrated that dairy products containing protein–modified matrices increase Lactobacillus and Bifidobacterium abundance, improve cytokine profiles (e.g., elevated interleukin–10 [IL–10] and reduced tumor necrosis factor–alpha [TNF–α]) (Santiago–López et al., 2018), and enhance systemic antioxidant markers, confirming the translational potential of these functional dairy systems.

Microbiota modulation

The human gut microbiota, often referred to as a “forgotten organ,” comprises approximately 95% of all the cells in the human body and contains an estimated 10¹¹–10¹² CFU/g of intestinal content (Fucarino et al., 2022; Yao et al., 2021). The gut microbiota plays a vital role in maintaining epithelial barrier function and supporting metabolic and biochemical activities. Probiotics have been shown to promote intestinal eubiotics through various processes, including the synthesis of SCFAs, enhancing gut barrier integrity, competitive exclusion of pathogens, and modulation of immunological responses (Gao et al., 2022).

Protein conjugates modulate gut microbiota by enhancing probiotic adhesion and supplying selective metabolic substrates. MTGase–mediated casein conjugates strengthen hydrophobic and electrostatic interactions with intestinal epithelial cells, improving colonization and persistence (Gao et al., 2022; Wang et al., 2020). In parallel, the gradual hydrolysis of protein–carbohydrate conjugates releases short peptides and reducing sugars that act as prebiotic–like substrates, promoting the growth of Bifidobacterium and Lactobacillus while suppressing less favorable taxa through nutrient competition (Abdeen et al., 2024). Human studies support these mechanisms, showing an increased fecal abundance of beneficial genera and a favorable Firmicutes–Bacteroidetes ratio in subjects consuming protein–modified dairy matrices (Vivek et al., 2023). Thus, protein conjugates serve as both protective carriers and active modulators of microbiota composition.

Freeze–drying L. plantarum with whey protein and sodium alginate enhanced the survival of probiotics compared to spray drying (Liu et al., 2019). This treatment process is crucial for maintaining the viability of probiotics during processing conditions. Further supporting this, studies on probiotic cheese highlight the positive effects of probiotics on gut microbiota. For example, supplementation with goat cheese containing L. rhamnosus EM1107 in mice challenged with S. enteritidis decreased Salmonella colonization while promoting the development of beneficial bacteria (Rolim et al., 2021). Thus, the combination of protective carriers, such as whey protein and sodium alginate, during freeze–drying effectively enhances probiotic viability, which, as evidenced in probiotic–enriched cheese studies, translates into improved gut microbiota balance and pathogen suppression.

Studies have also investigated the various benefits of probiotics in ice cream, apart from the encapsulation process. Chaikham and Rattanasena (2017) studied ice cream complemented with L. acidophilus LA–5 and L. casei 01 probiotic flora, providing the update about the modulation effect of the product against colon microbiome, which led to an increase in helpful secondary metabolites (e.g., butyrate, acetate, lactic acid, and propionate) because of the supplementation. This demonstrates how probiotics in dairy products can contribute to the production of metabolites that enhance digestive health.

Immune response and regulation in dairy foods

The effectiveness of Lactobacillus and Bifidobacterium probiotics in dairy products supports their role in enhancing the immune system, thereby reducing the risk of inflammatory diseases. When incorporated into dairy products with protein conjugates, these probiotics often exhibit improved stability in dairy products; they are better protected during digestion when combined with protein conjugates, such as whey protein, which enhances and regulates gut homeostasis and immune functioning (Liu et al., 2019).

Protein conjugates enhance probiotic–host immune interactions by stabilizing cell–surface proteins and facilitating mucosal adhesion, which amplifies recognition by dendritic cells and epithelial receptors (e.g., toll–like receptors 2 and 4 (TLR2 and TLR4) (Gao et al., 2022). This interaction modulates cytokine profiles, increasing anti–inflammatory mediators (IL–10) while reducing pro–inflammatory markers (TNF–α and interleukins–6 [IL–6]) (Kariyawasam et al., 2021). Some conjugates also release bioactive peptides with direct immunomodulatory activity, such as opioid–like or angiotensin–converting enzyme (ACE) inhibitory peptides (Han et al., 2020). Human intervention studies with dairy–based protein conjugates have reported reductions in systemic inflammation and enhanced mucosal immunity, supporting their translational relevance.

A study conducted on fermented milk samples produced from a blend of Lactobacillus and Bifidobacterium strains demonstrated immune–enhancing effects. Consuming fermented milk containing the mentioned probiotics has been linked to increased immune markers, such as immunoglobulin A (IgA), IL–6, and IL–10, which are crucial for enhancing animal mucosal immunity (Santiago–López et al., 2018). The findings of this study provided information on how probiotic fermentation can more favorably modulate immune responses and contribute to the overall health benefits. Additionally, Mai et al. (2021) found that probiotic fermented milk has shown potential in alleviating digestive– and respiratory–related problems in young children. The findings indicated that L. casei Shirota in probiotic milk drastically reduced constipation, diarrhea, and respiratory infections in children aged 3–5 years.

These findings suggest that adding protein conjugates to dairy–based probiotics enhances their capacity to modulate the immune system. As shown in Figure 4, protein conjugation enhances probiotic function, including improved gastric survival, targeted intestinal delivery, enhanced mucosal adhesion, and synergistic bioactivities (e.g., antioxidant and anti–inflammatory effects). Compared to free probiotics, conjugated probiotics more effectively modulate gut microbiota, strengthen the intestinal barrier, and support immune regulation.

Figure 4. Schematic illustration of the protective role of protein–polysaccharide conjugates in improving probiotic functionality. Native probiotics face challenges, such as poor survival in gastric and bile conditions, low intestinal adhesion, and reduced bioactivity during processing and storage. Source: Hao et al. (2025); Jang et al. (2024); Latif et al. (2023); Lim et al. (2024).

Furthermore, fermented milk with probiotic strains provides health benefits by producing bioactive peptides, including ACE inhibitors and antioxidative peptides. Production of high gamma–aminobutyric acid has been observed, with up to 5.5 g/L when S. thermophilus is present and up to 8.3 g/L when co–cultured with L. rhamnosus in casein hydrolysate–enriched milk (Han et al., 2020). Bifidobacterium and Lactobacillus probiotics are essential components of the human gut microbiota, playing a crucial role in restoring intestinal barrier function and enhancing immune function (El Jeni et al., 2024).

Although probiotic–protein immunomodulatory interactions remain underexplored, available evidence suggests that they are strain– and matrix–dependent. Recent studies have demonstrated that Lactobacillus strains can stimulate regulatory T cells to produce IL–10 and transforming growth factor–beta (TGF–β), increase IL–6 secretion in a TLR–2–dependent manner, and induce clonal expansion of IgA–producing B cells. Muhammad et al. (2026) showed that multi–species synbiotic supplementation, containing six probiotic species with inulin and FOS, significantly increased serum IL–10 levels and fecal SCFAs in adult men with dyslipidemia after 12 weeks. This highlights the need for systematic comparisons across probiotic strains and protein or prebiotic matrices to identify formulations with targeted immunomodulatory effects.

Bioavailability enhancement of probiotics and bioactive peptides

Protein conjugates play a crucial role in enhancing the bioavailability of probiotics in dairy products. They are commonly referred to as wall materials, including carbohydrates (e.g., starch and maltodextrin) and proteins (e.g., casein and whey protein). The absence of these wall materials can highlight the viability of probiotics during spray drying, resulting in a complete loss of viability of probiotics (Liu et al., 2019). Binary systems, which utilize proteins and polysaccharides to form conjugates, provide better protection for probiotic systems (Deng et al., 2025).

Protein conjugates enhance bioavailability by forming protective matrices that delay gastric degradation and facilitate targeted intestinal release (Mao et al., 2022; Wu et al., 2021b). Cross–linked proteins maintain probiotic viability until the colon while simultaneously enhancing solubility and uptake of associated bioactive peptides (Sbehat et al., 2022). Once released, peptides can cross epithelial barriers more efficiently because of improved resistance against proteolysis (Lemaire et al., 2021). Clinical studies using conjugated whey proteins in fermented dairy products have shown higher plasma levels of bioactive peptides and improved metabolic outcomes, underscoring the connection between structural protection and functional absorption (Gu et al., 2024; Huang et al., 2024).

Research has demonstrated that spray drying L. zeae LB1 with a combination of Arabic gum and sodium caseinate results in higher survival proportions at pH 2.0, compared to using sodium caseinate alone (Liu et al., 2016). Probiotics face stress in handling processes, storage, and digestive conditions, but their survival is critical for efficacy. The most common international standards on fermented products require a minimum of 10⁷ CFU/g for L. acidophilus and 106 CFU/g for B. bifidum in fermented milk, with some specific variations in some countries (Stuivenberg et al., 2022). Ensuring probiotic viability through protein conjugate encapsulation helps to meet these regulatory requirements.

Protein–probiotic conjugates enhance probiotic stability during food processing, storage, and GI transit, although their effectiveness depends strongly on the protein matrix and conjugation strategy used. Plant– and dairy–based proteins, including rice, pea, whey, and soy protein isolates combined with inulin, have shown improved conjugation efficiency and protection of Lactobacillus reuteri against thermal and storage stresses, with rice and whey protein systems often providing superior stability (Mudgil et al., 2024b). Emerging technologies, such as 3D printing coupled with freeze–drying, further increase survival, achieving up to 96–98% viability of Lactiplantibacillus plantarum during extended storage and digestion (Yoha et al., 2021). In addition, highly resilient strains, such as bacillus coagulans, maintain over 99% viability under severe processing conditions, while alternative proteins, such as Spirulina isolates, show promising lyoprotective effects, outperforming whey protein under certain conditions (Maity et al., 2021). Consquently, selecting appropriate protein carriers and conjugation methods is critical for enhancing probiotic stability in functional food applications.

Antioxidant properties

Conjugation of milk carbohydrates with proteins through the Maillard reaction enhances protein functional properties, resulting in more heat–stable, soluble, and emulsified products (Zhang et al., 2020). Additionally, this conjugation enhances antioxidant activity by forming melanoidin and heterocyclic compounds, as well as reducing ketones, thereby improving the health–promoting properties of dairy products (Wu et al., 2021b). Specifically, when WPI is heated with reducing sugars, the Maillard reaction enhances its reducing power and scavenging capacity, improving its antioxidant properties (Zhang et al., 2020). Additionally, a study has shown that the synergistic effect of ascorbic acid enhances the survival of L. acidophilus in yogurt. At the same time, enzyme–based scavengers, such as glucose oxidase, have been found to increase B. longum counts by up to 40% (Gupta, 2024).

Accordingly, protein conjugation stabilizes the food matrix while introducing antioxidant–active groups, particularly reductones and melanoidins, which scavenge reactive oxygen species (ROS) and chelate pro–oxidant metal ions (Wu et al., 2021b). This dual action mitigates oxidative stress in the gut, protecting encapsulated probiotics while simultaneously delivering systemic antioxidant benefits to the host (Noori et al., 2025). Clinical investigations corroborate these effects, demonstrating that dairy products enriched with protein conjugates improve redox balance, as evidenced by increased total antioxidant capacity and decreased lipid peroxidation in human subjects (Lemaire et al., 2021; Mudgil et al., 2024a).

Moreover, synbiotic yogurts have gained popularity because of their antioxidant, anti–inflammatory, and antihypertensive properties, which have a positive impact on the overall health. For example, synbiotic formulations containing L. brevis KU200019 and FOS have exhibited antioxidant, ACE–inhibitory, and immune–modulating effects in dairy products (Kariyawasam et al., 2021). Yogurt containing L. rhamnosus and B. lactis improves gut motility and relieves irritable bowel syndrome (IBS) (Olson and Aryana, 2022). Similarly, cheese fortified with L. casei and L. plantarum has been linked to reduced blood pressure and body mass index (BMI) (Shah et al., 2024). Additionally, ice cream enriched with L. acidophilus LA–5 and L. casei 01 increased the modulation of SCFAs production and promoted beneficial gut microbiota (Chaikham and Rattanasena, 2017). Another probiotic ice cream containing L. lactis NZ1330 has been shown to reduce Immunoglobulin E (IgE) levels and allergic responses in mice (Vasiee et al., 2020). Therefore, the antioxidant properties of dairy probiotic products suggest that their presence supports the health–promoting features of these products, as probiotics in them have been observed to remain qualitatively intact during GI transit. Beyond direct food applications, protein conjugates have found innovative uses in active packaging systems. For instance, whey protein–polyphenol conjugates incorporated into carboxymethyl cellulose films demonstrate enhanced vapor barrier properties, tensile strength, and intelligent pH–sensing capabilities for monitoring of freshness (Zhao et al., 2023). This innovative approach offers potential for extending the shelf life of dairy products while preserving probiotic viability.

Challenges Associated with Protein Conjugates in Dairy Foods

Hussain et al. (2021) postulated that the use of protein conjugation in dairy probiotics presents both problems and opportunities that must be addressed carefully prior to its application in any domain. Among the discrepancies are the technological challenges that have become most common. The issues of stability and the need for efficient delivery systems add to the complexity of conjugation methods, making it more challenging to incorporate them into protein conjugates containing probiotic dairy products. Another significant barrier to the market’s widespread acceptance of protein–conjugated probiotics is the complex regulations governing the clearance process and safety investigations for novel compounds (Cerk and Aguilera-Gómez, 2022). Consumer acceptance and market perception also predispose the success of protein–conjugated probiotics. Purchase decisions are influenced by a product’s efficacy, safety, and health benefits, as evidenced by a study on the development of the Maillard reaction in UHT milk under varying storage temperatures and thermal cycles (Sunds et al., 2018).

Technological challenges

Probiotics face significant challenges in maintaining viability and functionality throughout the manufacturing, processing, and storage of dairy products. The main factors affecting the survival and stability of probiotics in dairy foods are low temperatures, pH levels, and the composition of the probiotics (Deng et al., 2025; Mazhar et al., 2024). Thermal destabilization and low microbial growth present crucial challenges in dairy product manufacturing, as high–protein dairy matrices, such as evaporated milk, undergo structural changes under heat stress that compromise stability and probiotic viability. Wu et al. (2021a) investigated the modification of milk proteins via glycation via the Maillard reaction to enhance functionality by improving heat stability, eliminating the need for harmful catalysts, and optimizing the performance of dairy proteins during thermal processing.

Furthermore, whey proteins have relatively weak emulsifying properties, leading to instability, turbidity, and phase separation in acidic dairy products, especially during storage and heat exposure (Dıblan et al., 2024). Co–encapsulation with prebiotics serves as a potential strategy to enhance probiotic survival by protecting the cells from environmental stress during processing, storage, and digestion (Rashidinejad et al., 2022). Therefore, addressing these technological challenges through advanced protein conjugation and co–encapsulation strategies is a key to improving the stability and functionality of probiotics in dairy applications.

Regulatory barriers and safety concerns of protein conjugation

The practical applicability of protein–conjugation in dairy probiotics is linked to specific regulatory challenges. The US Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) are regulatory agencies that require comprehensive safety data, including toxicity and allergen tests, before these products are permitted for human consumption, as reported by Cerk and Aguilera–Gómez (2022) and Zavišić et al. (2023). Strict rules must be backed by solid scientific research when labeling products to make health claims, such as those that boost immunity. Although both EFSA and FDA have established regulations for probiotics and food additives, a gray area remains regarding protein–probiotic conjugates with exception of the study reported by Awaluddin et al. (2025), in which the study confirmed that synbiotic systems exhibit superior biological functionality, compared with probiotic or prebiotic alone, reflecting coordinated regulation of gut structure, nutrient bioavailability, and host performance. Manufacturing these conjugates at a larger scale while maintaining consistent quality and stability can be challenging, and additional steps may be required if the ingredient is considered novel (Spacova et al., 2023).

Moreover, while protein conjugation can enhance probiotic stability and functionality, one critical safety concern is the unintended formation of AGEs, which have been implicated in various metabolic and chronic diseases. The accumulation of AGEs in serum induces oxidative stress and activates the chromogranin A–uncoupling protein 2–glucose transporter 1 pathway, contributing to diabetes–related complications (Sun et al., 2023). Beyond diabetes, dietary AGEs exacerbate inflammation and oxidative stress, representing risk factors for chronic liver diseases. Another study confirmed that higher intake of AGEs, particularly Nε–(carboxymethyl) lysine and methylglyoxal–derived hydroimidazolone–1, were positively correlated with the incidence of fatty liver disease (Xie et al., 2023). To counteract these risks, several strategies are explored to inhibit the formation of AGEs. Approaches include blocking early glycation intermediates, preventing protein cross–linking, chelating metal ions, and suppressing AGE to their receptor (AGE–RAGE) signaling through antioxidant and anti–inflammatory pathways. Potential interventions involve glycation inhibitors (e.g., aminoguanidine and pyridoxamine), AGE breakers (e.g., alagebrium chloride), metal chelators, and natural phytochemical antioxidants (Gu et al., 2024). Excessive heating causes browning, loss of amino acid, and formation of AGE; thus, Bi et al. (2023) recommended 70°C for 15 s to preserve quality and nutrition in dairy systems.

Protein conjugation, while promising for improving stability and functionality, raises critical concerns regarding allergenicity Spacova et al. (2023). Structural modifications can mask epitopes and sometimes reduce IgE–binding, but uncontrolled conjugation reactions may expose new epitopes or generate neoallergens, increasing sensitization risks (Vasiee et al., 2020). Enzymatic methods, such as MTGase, further complicate this issue by altering the conformational epitopes of caseins and whey proteins, which can unpredictably affect immune recognition (Chen et al., 2020). Evidence to date is dominated by in vitro IgE–binding assays and rodent models, both of which have limited predictive value for human allergic responses (Cerk and Aguilera-Gómez, 2022). This lack of translational data creates uncertainty for regulatory assessment, as agencies, such as EFSA and FDA, mandate rigorous evaluation of allergenicity for novel protein modifications, including structural, digestibility, and clinical immunogenicity data. Without systematic human studies, the allergenic safety of conjugated milk proteins remains unresolved, highlighting the urgent need for targeted allergenicity assessments before such technologies can be safely applied in probiotic dairy foods.

Future perspectives and research needs in protein conjugation–based dairy products

The advent of protein conjugates in dairy products presents both opportunities and challenges from consumer and technological perspectives, as summarized in Table 4. They enhance probiotic survival, emulsification, and solubility, but also introduce off–flavors and bitterness if the Maillard reaction is not carefully controlled (Sunds et al., 2018). Consumer resistance to genetically modified organisms (GMOs), particularly in regions such as the European Union (EU), supports the use of non–GMO approaches, such as protein conjugation, to enhance acceptance (Börner et al., 2019; Hussain et al., 2021). As gut health remains a primary interest in probiotic consumption, improving formulations to show that clear health benefits can enhance consumer appeal (Gao et al., 2021; Rashidinejad et al., 2022). Therefore, optimization of the Maillard reaction is also crucial to avoid negative impacts on the appearance and flavor of products (Wu et al., 2021b).

Protein conjugates offer functional benefits, such as enhanced probiotic delivery; however, they may negatively affect color and flavor (Deng et al., 2025; Wu et al., 2021b). Incorporating postbiotics, including those derived from L. acidophilus, further enhances immune responses. Using strains such as L. reuteri and other fermented probiotic derivatives can strengthen these effects (Garnier et al., 2019). Additionally, probiotic–based exopolysaccharides enhance the rheological properties of dairy products, improving their texture, flavor, and consumer appeal while contributing to health benefits (Ali et al., 2023; Zhao and Liang, 2023). Thus, future studies are expected to provide information on how these upcoming industrial processes may affect probiotic dairy foods in a practical setting.

Future research should combine detailed mechanistic investigations, such as molecular signaling, microbial adhesion, and bioactive peptide release, with well–designed human clinical trials to validate the effects of protein conjugates on probiotic functionality, gut microbiota modulation, and immune responses within real–world dairy matrices. Further cost–benefit analysis of protein conjugation is required to assess its economic feasibility and guide industrial–scale implementation while balancing functional efficacy and product quality.

Study Limitations

Although this review provides an integrated overview of protein conjugation strategies and their impact on probiotic functionality in dairy systems, several limitations must be considered. First, most of the available evidence is derived from in vitro experiments and animal models, which do not fully capture the complexity of human digestive and immune responses. For example, allergenicity assessments rely heavily on rodent models and IgE–binding assays, both of which exhibit limited predictive value for human outcomes (Cerk and Aguilera–Gómez, 2022; Vasiee et al., 2020). Similarly, most studies evaluating thermal stability, GI tolerance, or encapsulation performance examine individual probiotic strains, despite clear strain–dependent variability in conjugation efficiency and functional response (Li et al., 2025; Liao et al., 2021; Mudgil et al., 2024b). Second, variations in processing conditions, such as temperature, RH, pH, and ionic strength, that affect reproducibility and hinder cross–study comparison. Lack of standardized protocols for the Maillard reaction, enzymatic, or chemical conjugation leads to difficulties in identifying optimal conditions and determining scalability for industrial applications (Gao et al., 2021; Wu et al., 2021b). Additionally, most current reports focus on small–scale laboratory processes, with limited evaluation of pilot– or industrial–scale production, limiting insights into cost, energy demand, and process robustness (Parhi et al., 2024). Moreover, the potential formation of AGEs during the Maillard conjugation presents unresolved safety concerns. Although the existing research highlights correlations between dietary AGEs and inflammatory or metabolic disorders (Sun et al., 2023; Xie et al., 2023), there is still limited research that measures AGE formation in conjugated dairy systems or examines the potential long–term exposure risks in humans. Finally, the regulatory landscape for protein–probiotic conjugates remains underdeveloped. Limited human clinical trials, insufficient allergenicity data, and unclear classification frameworks restrict the translation of these technologies into commercial dairy products (Cerk and Aguilera–Gómez, 2022; Spacova et al., 2023). Addressing these limitations requires coordinated efforts to develop standardized methodologies, conduct well–designed human studies, and establish regulatory guidance specific to conjugated protein systems.

Conclusions

Protein conjugation, through processes such as the Maillard–type and enzymatic cross–linking, has demonstrated considerable potential in enhancing the functionality and viability of probiotics in dairy systems. These conjugates act as dual–function systems, providing a protective barrier for probiotic cells under adverse conditions, such as heat, acidity, and bile salts, while simultaneously improving texture, emulsifying properties, and the overall stability within dairy matrices. Consistent findings across various applications continue to show improvements in encapsulation efficiency, thermal tolerance, and water–holding capacity, which together support the broader integration of conjugated systems in both fermented and nonfermented dairy products.

Despite these advantages, broader application remains limited by several factors, including the need for tighter control of the Maillard reaction conditions to prevent excessive browning and the formation of undesirable compounds, along with the continued challenge of managing potential sensory changes and navigating complex regulatory frameworks related to food safety and functional claims. Consequently, future research on protein conjugation should prioritize the refinement of reaction conditions, particularly through optimization of the Maillard reaction and enzymatic pathways to improve reproducibility, minimize undesirable end products, and enhance safety. In addition, exploring novel protein and carbohydrate sources with promising functional and nutritional attributes may further expand the applicability of conjugates across diverse food systems.

Personalized functional dairy foods represent a promising direction, emerging from the integration of gut microbiome profiles, metabolic and immune biomarkers, and indicators of gut–barrier function. When combined with predictive modeling approaches, including machine–learning (ML)–based diet–microbiome response tools, these developments open the possibility for designing protein–probiotic conjugates that align more closely with individual health objectives, such as metabolic regulation, GI tolerance, and immune modulation. Within this framework, protein conjugation offers opportunities to improve nutrient interactions, modulate allergenicity, and selectively influence host–microbiota dynamics.

However, translating these advances into practical applications require comprehensive cost–benefit evaluations and well–designed human clinical studies to verify efficacy and safety beyond laboratory and animal evidence. At the same time, successful commercialization depends on achieving industrial scalability through environmentally responsible and economically viable processing strategies, supported by artificial intelligence (AI)–driven and omics–based formulation tools that strengthen process robustness and accelerate innovation. Ultimately, long–term progress depends on adherence to international regulatory frameworks to ensure that functional claims are scientifically substantiated and that consumer safety is maintained consistently.

Data Availability

The data are available upon request.

Mandatory Disclosure on Use of Artificial Intelligence

The authors declare that no AI-assisted tools were used in the preparation of this manuscript. All references have been manually verified for accuracy and relevance.

Author Contributions

Conceptualization, writing–original draft preparation, and writing–review and editing: Nareekan Chaiwong, Auengploy Chailangka, Idris Kaida Zubairu, Tri Indrarini Wirjantoro, Pavalee Chompoorat Tridtitanakia, and Mohsen Gavahian; writing–review and editing: Juan Manuel Castagnini and Mahsa Majzoobi; supervision, conceptualization, funding acquisition, and writing–review and editing: Yuthana Phimolsiripol. All authors have read and agreed to the published version of the manuscript.