Probiotic Dairy Innovations: Exploring Buffalo Milk Potential for Food Product Development

ABSTRACT

Buffalo milk has emerged as a promising substrate for probiotic dairy innovations due to its distinctive nutritional profile and diverse microbial community. This review critically examines the current knowledge on buffalo milk and its potential applications in developing functional probiotic dairy products. Buffalo milk offers a rich matrix of bioactive components, with higher levels of fat, vitamin A, and biotin, along with comparable calcium content but lower sodium levels relative to cow milk. These compositional differences may have implications for calcium absorption and retention. Lower sodium levels support calcium balance, whereas higher saturated fat content may reduce calcium absorption efficiency, highlighting the need for further study. The review explores the microbial diversity of buffalo milk, emphasizing the prevalence of lactic acid bacteria and other probiotic candidates, and discusses their suitability for use in fermented dairy products such as yoghurt, kefir, and cheese. Innovative processing strategies, including microencapsulation and non‐thermal technologies, are assessed for their roles in enhancing probiotic survival and product quality. Additionally, global regulatory frameworks and safety considerations for probiotic dairy products are outlined, along with advanced analytical approaches such as metagenomics and metabolomics for product evaluation. Despite its promising attributes, significant knowledge gaps remain, particularly regarding probiotic strain performance, nutrient bioavailability, consumer acceptance, and clinical validation of health benefits specific to buffalo milk‐based products. Addressing these gaps through targeted research will support the development of high‐value, health‐promoting functional dairy products derived from buffalo milk, particularly in regions where buffalo farming contributes to sustainable food systems.

1. Introduction

Food plays a fundamental role in maintaining health and wellbeing by providing essential nutrients, energy needed by our bodies for growth, maintenance, and repair, while supporting physiological functions. Beyond basic nutrition, food serves as a carrier for functional ingredients, including probiotics, prebiotics, phytosterols, omega‐3 fatty acids, minerals, vitamins, and bioactive peptides (Kaur and Das 2011; Shahidi 2009). Although there is no universal definition, functional foods are generally understood as foods or ingredients that, when consumed regularly in normal dietary amounts, provide health benefits or reduced disease risk beyond basic nutrition (Granato et al. 2020). Growing consumer awareness of health benefits has led to an increasing demand for functional foods enriched with probiotics and bioactive compounds. In response, the food industry has expanded its focus on developing such products (Turkmen et al. 2019). This shift has contributed to rapid market growth, with the sector expected to reach USD 386.2 billion by 2033, led by dairy products exhibiting a growth rate (CAGR) of 5.45% during 2025–2033 (IMARC 2025). Regulatory frameworks now govern health‐related claims on functional food labels to ensure consumer transparency and product efficacy (Domínguez Díaz et al. 2020; Martirosyan et al. 2021).

Milk is among the top five agricultural commodities globally in terms of both quantity and value, with world milk production reaching 930 million tonnes in 2022 (FAO 2022). Milk is a nutrient‐dense food, providing essential protein, fat, minerals, and lactose (Roy et al. 2020). Dairy products, such as milk, yoghurt, and cheese, form an integral part of diets in both Western and developing nations, contributing significantly to nutrition and overall health (Hettinga and van Valenberg 2017; Pereira 2014). In Australia, the National Health and Medical Research Council (NHMRC) classifies dairy products as core food groups. They provide key sources of calcium and high‐quality protein, with recommended daily intakes of 2–4 servings across various age and gender groups (NHMRC 2013). The dairy industry has leveraged milk's natural functionality by incorporating probiotics into products like yoghurt, cheese, kefir, and ice cream, thereby creating a range of functional dairy foods (Martins et al. 2017; Ortiz et al. 2017).

Raw milk supports a diverse microbiota, comprising bacteria, fungi, yeasts, and viruses (Quigley et al. 2013; Wochner et al. 2018; Akinyemi et al. 2021). This microbial community includes beneficial microorganisms like Lactobacillus and Bifidobacterium species. It also contains potentially harmful strains, including spoilage organisms (Pseudomonas, Clostridium, Bacillus, and other spore‐forming or thermoduric microorganisms) and pathogens (Listeria, Salmonella, Escherichia coli, Campylobacter, and mycotoxin‐producing fungi). Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al. 2014). Probiotics play a crucial role in improving gut health, boosting immunity, and providing metabolic benefits.

Probiotic‐based functional foods have experienced a significant rise in global consumption due to their diverse health benefits (Champagne et al. 2018). Functional dairy products, including probiotic‐enriched milk, yoghurt, and cheese, can contain bioactive components such as omega‐3 fatty acids, vitamins, minerals, and plant extracts. The global probiotics market, valued over $34.1 billion in 2020, is projected to double by 2030 driven by increasing consumer demand (Allied Market Research 2023). Probiotics exert their effects through mechanisms such as colonization resistance, immunomodulation, and metabolic activities, including cholesterol assimilation and vitamin production (Soccol et al. 2010). To be classified as probiotics, microorganisms must meet specific in vitro and in vivo criteria. These include being non‐pathogenic, producing antimicrobial compounds, resisting gastric acid and bile, and tolerating industrial processing conditions (Al‐Saeed 2017; Oikonomou et al. 2020). Additionally, adherence to gut epithelial tissue is desirable, as it may enhance colonization and functional efficacy (Morelli and Capurso 2012). Although Lactobacillus and Bifidobacterium remain the most widely used probiotic genera, recent studies have expanded research to identify novel strains with strain‐specific benefits. Fermentation of milk by its microbiota enhances the nutritional and microbiological properties, producing antimicrobial peptides and other bioactive compounds (Akinyemi et al. 2021). The search for probiotic strains with industrial and health relevance continues to be a key focus in dairy research (Sakandar and Zhang 2021). However, the viability and stability of probiotics remain significant technical challenges in developing next‐generation probiotic foods (Wilkinson 2018). In response to these challenges, emerging strategies such as microencapsulation, nanotechnology‐based delivery systems, and non‐thermal processing techniques are being applied to improve probiotic survival in dairy matrices (Akdeniz and Akalın 2022; Aliabbasi and Emam‐Djomeh 2024). These innovations are particularly relevant as research expands into non‐bovine milk sources, such as buffalo milk, which offers a distinct nutritional matrix and microbial compatibility for novel probiotic product development.

Although extensive research has been conducted on probiotic development in cow milk, buffalo milk remains relatively underexplored. Despite its unique composition, which can enhance probiotic stability and functionality, research on buffalo milk‐derived probiotics is limited (Vargas‐Ramella et al. 2021). Key challenges include its regionalized production, compositional variability that complicates standardization, and lower consumer demand for non‐cow milk probiotic products. Nevertheless, buffalo milk offers significant potential due to its higher protein, fat, and bioactive compound content. Its favorable mineral profile may also help support probiotic survival and improve calcium retention. Recent research efforts have begun to address this gap by isolating and characterizing novel lactic acid bacteria (LAB) from buffalo milk fermentations (Ginting et al. 2025), signaling a growing scientific interest in buffalo milk's probiotic potential. These investigations have uncovered new strains with potential functional benefits, highlighting buffalo milk as a promising medium for microbial innovation. Beyond its nutritional and microbiological advantages, buffalo milk also plays a critical role in sustainable food systems, particularly in climate‐vulnerable and low‐resource settings. In South Asia, which produces over 96% of global buffalo milk, buffaloes are vital for rural livelihoods and food security, thriving in hot climates and converting poor‐quality forage into nutrient‐rich milk. In Latin America and the Mediterranean, buffalo farming supports small‐scale producers and traditional high‐value dairy products, improving land use efficiency and income stability (FAO 2023; Hernandez and Bonilla‐Landaverry 2025). This review examines the properties, processing, and products of buffalo milk, emphasizing its microbial diversity, particularly LAB, and their probiotic potential in fermented dairy products. The overview of key themes covered in this review has been illustrated in Figure 1. By addressing existing research gaps and leveraging buffalo milk's unique microbial diversity, researchers can unlock new opportunities for probiotic applications and enhanced dairy product development. As consumers increasingly seek health‐promoting foods, buffalo milk's probiotic effects have been linked to potential reductions in the risk of various diseases (Vargas‐Ramella et al. 2021). This underscorescontinued research to validate and harness these benefits. Additionally, strategic approaches to improve market accessibility and diversify functional food options derived from buffalo milk are discussed.

FIGURE 1.

Overview of key themes covered in this review.

2. Characteristics and Applications of Buffalo Milk

2.1. Chemical Composition of Buffalo Milk

Cow milk remains the most widely consumed milk globally, accounting for 81% of the total milk production in 2019. Buffalo milk follows at 15%, then goat milk (2%), sheep milk (1%), and camel milk (0.4%) (OECD & FAO 2019). Table 1 presents a comparative analysis of the chemical composition and nutritional value of these milk sources.

TABLE 1.

Comparative chemical composition and nutritional value of milk from different animal species.

Compound	Buffalo milk	Cow milk	Sheep milk	Goat milk
Milk density (g/cm3)	1.037	1.031	1.039	1.032
Chemical composition
Dry matter (%)	16.67	13.32	18.05	13.56
Solid non‐fat (%)	10.09	9.13	11.24	8.95
Protein (%)	5.20	3.32	5.5	3.4
Casein (g/100 mL)	3.20	2.80	4.46	2.81
Fat (%)	8.80	4.17	6.82	4.61
Lactose (%)	5.36	4.7	5.1	4.4
Total ash (%)	0.90	0.8	0.58	0.85
Energy (kJ/kg)	4054	3730	5932	3018
pH	6.6	6.65–6.71	6.51–6.85	6.5–6.8
Acidity (%)	0.12	0.13	0.14	0.12
Amino acids (g/100 g protein)
Threonine	5.71	4.5	4.4	5.7
Cysteine	0.59	0.6	0.9	0.6
Valine	8.28	4.8	6.4	5.7
Methionine	1.99	1.8	2.7	3.5
Isoleucine	5.71	4.2	4.2	7.1
Leucine	9.79	8.7	9.9	8.2
Tyrosine	3.86	4.5	3.8	4.8
Phenylalanine	4.71	4.8	4.3	6.0
Aspartic acid	7.13	7.8	6.5	7.4
Serine	4.65	4.8	3.4	5.2
Glutamic acid	21.4	23.2	14.5	19.3
Proline	12.0	9.6	16.2	14.6
Glycine	1.93	1.8	3.5	2.1
Alanine	3.03	3.0	2.4	3.6
Histidine	2.73	3.0	6.7	5.0
Lysine	9.84	8.1	7.8	8.2
Cholesterol (mg/100 g of milk)	17.96	90	43.2	42.1
Fatty acids (%)
C4:0	4.18	4.2	2.57	2.03
C6:0	2.78	2.9	1.87	2.78
C8:0	2.98	2.9	1.87	2.92
C10:0	3.21	4.6	6.63	9.59
C12:0	3.92	2.6	3.99	4.52
C14:0	10.97	13.0	10.17	9.83
C16:0	30.17	28.8	25.1	24.61
C18:0	13.79	13.9	8.85	8.87
C18:1n‐9	25.17	—	—	—
C18:2n‐6	1.84	—	—	—
SFA	68.31	67.5	75.0	74.0
MUFA	28.32	30.3	39.0	36.0
PUFA	3.10	3.0	7.3	5.6
CLA	0.58	1.1	1.1	1.2
Minerals (mg/100 g)
Calcium	148	122	200	134
Phosphorus	107	119	158	121
Potassium	92	152	140	181
Magnesium	14	12	21	16
Sodium	37	58	58	41
Zinc	0.46	0.53	0.58	0.56
Iron	0.16	0.08	0.122	0.007
Copper	0.04	0.07	0.07	0.005
Manganese	0.07	0.02	0.006	0.003
Iodine	0.004	0.0021	0.014	0.002
Selenium	0.006	0.00096	0.003	0.001
Vitamin (mg/100 g)
Vitamin A a	69	46	146	185
Vitamin E a	0.19	0.21	—	0.03
Thiamin	0.05	0.05	0.08	0.068
Riboflavin	0.11	0.17	0.37	0.21
Niacin	0.17	0.09	0.416	0.27
Folate (µg)	6.15	4.91	6.96	0.82
Pantothenic acid	0.15	0.37	0.408	0.31
Vitamin B6	0.33	0.04	0.08	0.046
Vitamin B12	0.40	0.45	0.712	0.665
Biotin	13	2.0	0.93	1.5
Vitamin C a	2.5	0.09	4.16	1.29
Vitamin D	2.0	2.0	1.18	1.33

Abbreviations: CLA, conjugated linoleic acid; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids.

^aVitamin possesses antioxidant activity.

Source: Chemical composition (Rafiq et al. 2016; Vargas‐Ramella et al. 2021; Park et al. 2007); amino acids profile, vitamin, mineral content (Khan et al. 2019; Vargas‐Ramella et al. 2021); fatty acid profile (Mollica et al. 2021; Markiewicz‐Kęszycka et al. 2013; Vargas‐Ramella et al. 2021).

The composition of milk across species (e.g., cow, buffalo, goat, and sheep) is influenced by intrinsic factors such as species‐specific differences in milk components, host physiology, environmental conditions, and microbial interactions (Luo et al. 2024). Buffalo milk stands out for its superior richness in fat, protein, and essential amino acids compared to cow and goat milk, although it is slightly lower in protein content than sheep milk (Rafiq et al. 2016). Notably, buffalo milk contains a higher total casein content, composed of approximately α_{s 1}‐casein (32.2%), α_{s 2}‐casein (15.8%), β‐ and γ‐casein (36.5%), and κ‐casein (15.5%) compared to cow milk. Additionally, it has elevated levels of β‐lactoglobulin about 1.3 times that of α‐lactalbumin, which contribute to its excellent nutritional and functional properties (Bonfatti et al. 2013). The presence of higher levels of α‐casein and κ‐casein enhances its digestibility (Anusha Siddiqui et al. 2024).

The distinctive composition of buffalo milk makes it particularly beneficial for individuals requiring energy‐dense, high‐protein, and calcium‐rich foods, such as frail elderly individuals and residents of aged care facilities. Its superior amino acid profile, including higher concentrations of threonine, valine, leucine, lysine, and methionine, further elevates its nutritional value. Additionally, buffalo milk contains less than half the cholesterol of sheep and goat milk and approximately one‐fifth the amount found in cow milk (Rafiq et al. 2016). Buffalo milk's lipid composition also sets it apart. While providing more than twice the total fat content of cow milk, it maintains a similar ratio of saturated to monounsaturated to polyunsaturated fats, a characteristic not seen in sheep and goat milk. For example, its monounsaturated fatty acid (MUFA) content is slightly lower than cow milk, whereas its MUFA content surpasses that of sheep and goat milk. The larger fat globules in buffalo milk (60% within the 3.5–7.5 µm range) compared to cow milk (0.2–15 µm) enhance its creamy texture. Similarly, buffalo milk has larger casein micelles (70–200 nm) than cow milk (50–180 nm), which contribute to its improved textural properties and higher heat stability (Mejares et al. 2022). Buffalo milk has higher heat stability than cow milk, which is an advantage for dairy processing (Sharif et al. 2024). Beyond its macronutrient composition, buffalo milk is a rich source of essential micronutrients, particularly iodine and selenium, which support thyroid function and antioxidant defense. Although buffalo milk has a calcium level comparable to that of cow milk (Rafiq et al. 2016), its lower sodium concentration and higher total fat content may influence calcium absorption and retention in the body. Reduced sodium levels can enhance calcium bioavailability by lowering urinary calcium excretion, although the impact of higher fat content on absorption remains complex and warrants further investigation. High sodium intake is associated with increased urinary calcium excretion, potentially diminishing calcium retention (Sacco and L'Abbé 2016). Therefore, the lower sodium content of buffalo milk could support improved calcium balance. Conversely, the higher saturated fat content in buffalo milk may have a counteracting effect on calcium bioavailability, as evidence suggests that diets high in saturated fat may reduce calcium absorption efficiency (Teegarden 2005). However, direct comparative studies on calcium bioavailability from buffalo versus cow milk remain limited, highlighting an area for future research. Buffalo milk provides calcium levels comparable to cow milk, while offering higher amounts of vitamin A and biotin, nutrients essential for maintaining healthy vision and skin. These unique attributes position buffalo milk as an excellent candidate for probiotic dairy development and functional food innovations.

2.2. Buffalo Milk and Its Derived Products

Like cow milk, buffalo milk can be processed into a wide range of dairy ingredients and consumer products, including milk beverages, milk concentrates, powders, protein, butter, cream, yoghurt, cheese, and kefir. Buffalo milk can serve as a substitute for or be blended with cow milk to produce various dairy products. However, due to compositional differences, buffalo milk exhibits distinct nutritional, technological, and functional properties that influence product formulation and processing. These variations require modifications in processing conditions to ensure consistent and high‐quality buffalo milk‐based products (Mejares et al. 2022). For instance, buffalo milk has a higher viscosity than cow milk, although standardizing its fat content to 3% results in similar viscosity levels (Khalifa and Ghanimah 2013). Additionally, buffalo milk's larger fat globules and higher total solids content require adjustments in processing parameters—such as more intensive homogenization and careful heat treatments—to achieve stable emulsions and prevent quality defects in products (Mejares et al. 2022). These technological considerations are crucial for successfully translating buffalo milk's rich composition into consistent, consumer‐acceptable dairy products.

Numerous studies have explored the processing and quality assurance of dairy products made from cow milk (Chandan 2015). Conventional dairy products such as milk powders (Augustin and Clarke 2011), cheese (Johnson and Law 2010), and yoghurt (Moineau‐Jean et al. 2020) have been well documented. High protein dairy ingredients, including casein, milk protein concentrates (MPC) (Augustin and Clarke 2011; Bouvier et al. 2013), whey‐based ingredients (Huffman and de Barros Ferreira 2011), micellar protein concentrates (Rupp et al. 2018), and milk co‐precipitates, are produced using heat treatment, fractionation, concentration, and drying. Solubility and heat stability are critical factors in determining the functionality of these protein‐rich ingredients (Yousefi and Abbasi 2022). Rehydration properties are also key for protein powders, requiring optimization to improve their functionality in food applications (Fang et al. 2011; McSweeney et al. 2021).

Research has been conducted on processing and properties of dried buffalo milk dairy products. High‐quality buffalo milk powder can be obtained through spray drying (Mian et al. 2015; Borges et al. 2017). However, lactose‐hydrolyzed buffalo milk powders exhibit higher rates of browning and reduced hydration efficiency compared to standard milk powders (Lima de Paula et al. 2021). Co‐precipitates are high protein ingredients prepared by the simultaneous precipitation of casein and whey proteins using a combination of heat and acidification. Compared to milk protein isolates and concentrates, they have poor solubility but have applications in bread and bakery industries. Buffalo milk co‐precipitates have been prepared, and by optimization of process conditions, a maximum solubility of 31.5% and 42% were obtained for co‐precipitates with 76% and 80% protein, respectively (Gawande et al. 2022). Preparation of buffalo milk protein co‐precipitate using disodium hydrogen phosphate and trisodium citrate improved wettability, flowability, and heat stability (Gawande et al. 2022). The properties of buffalo milk co‐precipitates are in a similar range to cow milk protein co‐precipitates containing 79% protein with 39.6% solubility (Amila et al. 2022). MPC contain 40%–89% protein and have casein to whey ratios that are identical to milk and are prepared using processes including separation, heat treatment, ultrafiltration, and diafiltration. There are some challenges in producing buffalo milk concentrates due to its higher calcium and protein content. Buffalo milk protein concentrate (BMPC) powder generally exhibits lower solubility than those based on cow milk protein concentrate (CMPC) with similar protein content (Patil et al. 2019, 2018). For example, a comparison of BMPC powder (BMPC80; 96.5% total solids: 77.6% protein, 6.7% lactose, 9.99% ash, and 2.14% Ca) with that of CMPC powder (80% protein) indicated that buffalo milk BMPC80 had poorer solubility, heat stability, dispersibility, flowability, and porosity compared to CMPC made from cow milk (Mahadev and Meena 2020). The addition of sodium hexametaphosphate and sodium tripolyphosphate to the ultrafiltered retentate improved the solubility, fluidity, water binding, oil binding, foaming properties, and heat stability of BMPC powder (Shinde et al. 2021).

Buffalo milk is used in a variety of dairy products, including cheese, butter, paneer, and yoghurt (Anusha Siddiqui et al. 2024). Traditional fermented buffalo milk products such as meekiri (Sri Lanka), dadih and dahi (Southeast Asia), and lassie (India) contain indigenous probiotic bacteria including Enterococcus faecium, Lactiplantibacillus plantarum, and Limosilactobacillus fermentum (Abesinghe et al. 2020). However, further research is needed to explore probiotic fortification in these products. Recent work has started to explore such probiotic fortification. For example, Ginting et al. (2025) developed a probiotic ice cream by incorporating dadih, a traditional fermented buffalo milk. The product maintained viable probiotic counts (∼10⁶ CFU/g) and was well‐received in sensory trials. Such innovations illustrate buffalo milk's versatility in novel functional dairy foods. A comparison of the properties of probiotic yoghurt made from buffalo milk (17.1% total solids: 5.0% lactose, 4.1% protein, 7.9% fat, and 45.8 mM Ca) and fortified cow milk (16.8% total solids: 7.0% lactose, 4.6% protein, 4.1% fat, and 37.8 mM Ca) showed that both yoghurts had similar gel firmness. However, buffalo milk yoghurt displayed a weaker, more porous, and irregular network structure, which was further disrupted by large, unhomogenized fat globules and exhibited a higher degree of syneresis (Nguyen et al. 2014). They also observed that the starter cultures Streptococcus thermophilus and Bifidobacterium lactis Bb‐12 grew better in the fortified cow milk than in buffalo milk. An exception was Lactobacillus acidophilus La‐5, which showed similar growth in both milks (Nguyen et al. 2014). Others found that post‐acidification in buffalo milk beverage (5.1% fat, 4.28% protein) fermented with S. thermophilus, Lactobacillus bulgaricus LB340, and L. acidophilus was slower compared to cow milk beverage (3.52% fat, 2.88% protein), and further, the survival of probiotic bacteria, especially L. acidophilus, during in vitro gastrointestinal transit was higher in products made from buffalo milk (Simões da Silva et al. 2020). Kefir made from buffalo milk had higher water‐holding capacity and firmness than that made from cow milk, having better sensory properties as well (Gul et al. 2018). Furthermore, buffalo milk yoghurt fermented with selected probiotics can deliver additional health benefits. For instance, a 2025 study noted that buffalo yoghurt made with L. plantarum and B. lactis maintained high viable counts (>5 × 10⁵ CFU/g after 28 days) and exhibited enhanced antioxidant activity and angiotensin‐converting enzyme (ACE) inhibitory effects, underscoring its promise in developing heart‐healthy functional foods (Erturkmen 2025).

Buffalo milk has long been recognized as a suitable raw material for cheese production. However, cheese‐making protocols designed for one species may not always be directly applicable to another (Patel et al. 2024). Buffalo milk alone or in combination with cow milk is used to produce various cheeses. Traditionally, mozzarella cheese was made exclusively from buffalo milk, which differs from cow milk mozzarella in terms of sensory properties, composition, and microstructure (Kindstedt et al. 2010; Nguyen et al. 2017). The protein and fat in buffalo milk mozzarella are more resistant to oxidation than those in cow milk mozzarella (Balestrieri et al. 2002; Rinaldi et al. 2021). The use of different probiotic cultures, including L. acidophilus, L. plantarum, and Lacticaseibacillus rhamnosus, has been shown to enhance the functional and textural properties of mozzarella cheese, improving meltability and stretchability (Akarca et al. 2023). However, some studies suggest that while buffalo mozzarella has a higher calorific value and yield, cow mozzarella may have slightly better organoleptic quality (Ayoob et al. 2022). A study on the production of white soft cheese showed that cheese made from buffalo milk was more sensorially acceptable than those made with mixtures of buffalo milk and cow milk (Dimitreli et al. 2017). A comparison of cow and buffalo milk for fresh cheese production showed that buffalo milk yielded more cheese and enabled better nutrient recovery, both for acid‐ and rennet‐coagulated types (Patel et al. 2024). Additionally, buffalo milk is suitable for probiotic cheese production, such as Quark‐like cheese, which benefits from buffalo milk's high dry matter, fat, and protein content. Blending buffalo milk with cow milk at 25%–50% ratios has been recommended for producing fresh cheese with enhanced sensory and nutritional properties (Guneser and Aydin 2022). These findings highlight the potential for further innovation in buffalo milk‐based dairy products.

3. Comparative Analysis of Buffalo and Cow Milk Microbiota

The unique composition of milk fosters a distinctive microbial environment that enhances fermentation processes and increases its probiotic potential (Roy et al. 2020). Recent studies emphasize the critical role of milk microbiota in determining milk quality and safety (Fusco et al. 2020). Certain bacteria, particularly LAB, contribute to maintaining high‐quality standards in dairy products (Hou et al. 2020; Melchiorre et al. 2013). Raw milk harbors a diverse microbiota, including bacteria, fungi, yeasts, and viruses, making it essential to understand its microbial ecosystem for both public health and dairy innovation (Quigley et al. 2013).

LAB is naturally present in milk, but their composition depends on many factors such as milk origin, livestock diet, season, pasture altitude, processing, and hygiene practices (Terzić‐Vidojević et al. 2020). Key microbial groups in raw milk include LAB, coliforms, spoilage bacteria, and potentially harmful microorganisms (Quigley et al. 2013). Common microorganisms found in raw milk include species of Lactococcus, Lactobacillus, Pseudomonas, Micrococcus, Staphylococcus, and various yeast (Coelho et al. 2022). Table 2 lists the predominant LAB strains found in different animal milk sources, highlighting its potential for probiotic applications and dairy product development. Understanding the similarities and differences between cow and buffalo milk microbiota is crucial for dairy fermentation, probiotic development, and safety management in dairy products.

TABLE 2.

Comparative presence and functions of common lactic acid bacteria (LAB) strains in raw milk from different animal species (adapted from Quigley et al. 2013, with additional sources).

LAB strains	Animal sources	Primary functions	Notable differences
Lactiplantibacillus plantarum	Cow, Sheep, Buffalo	Fermentation, lactic acid production, pathogen inhibition, bio‐preservation, flavor enhancement	More frequently isolated in buffalo milk; higher acid and bacteriocin production in buffalo matrix (Huang et al. 2021)
Lactococcus lactis	Cow, Goat, Sheep, Buffalo	Acid production, texture and flavor development, cheese ripening, bacteriocin production	Present in both milks; higher exopolysaccharide (EPS) production potential reported in buffalo milk strains (Vargas‐Ramella et al. 2021)
Streptococcus thermophilus	Cow, Goat, Sheep, Buffalo	Yoghurt fermentation, EPS production, lactose metabolism, bioactive peptide formation	Found in both cow and buffalo milk; greater thermophilic LAB abundance in buffalo milk (Quigley et al. 2013; Vargas‐Ramella et al. 2021)
Enterococcus faecium	Cow, Goat, Sheep, Buffalo	Bacteriocin production, antimicrobial activity, flavor development, casein breakdown	Higher prevalence in raw buffalo milk; considered important for natural fermentation (Simões da Silva et al. 2020; Serrano et al. 2024)
Lacticaseibacillus casei	Cow, Sheep	Fermentation, enhancement of sensory properties, metabolic activity	Less frequently reported in buffalo milk; more dominant in cow milk‐derived products (Quigley et al. 2013)
Lactobacillus delbrueckii subsp. bulgaricus	Goat, Buffalo	Yoghurt fermentation, acid production, contribution to texture	Better viability and acidification kinetics in buffalo milk matrices compared to cow milk (Simões da Silva et al. 2020)
Lactobacillus helveticus	Goat	Bioactive peptide formation, cheese flavor enhancement, casein hydrolysis	Rare in buffalo milk; more often found in cow/goat‐derived milk (Garau et al. 2021)
Saccharomyces cerevisiae (yeast)	Buffalo, Cow	Enhances fermentation, contributes to aroma and flavor, assists LAB growth	Frequently co‐isolated in naturally fermented buffalo milk; enhances mixed culture stability (Abesinghe et al. 2020)

Note: Differences in microbiota profiles between cow and buffalo milk are influenced by intrinsic milk composition and traditional fermentation practices. Buffalo milk environments often support specific strains such as L. delbrueckii and E. faecium, enhancing their probiotic potential.

The microbiota of buffalo milk is diverse, including a wide range of bacteria, particularly LAB, similar to cow milk. These microbial communities play a crucial role in fermentation and preservation, significantly impacting probiotic development by influencing the stability, viability, and health‐promoting effects of probiotic strains used in fermented products. Understanding buffalo milk's microbial composition is essential for ensuring product safety and optimizing formulation strategies (Breyer et al. 2020). Despite its potential, the microbiological profile of buffalo milk, particularly the identification of beneficial bacterial strains suitable for use as starter cultures or probiotics, remains largely underexplored. A deeper investigation into this microbiota is critical for advancing both traditional and industrial dairy fermentation, paving the way for innovative functional probiotic products. Comparative studies have begun to reveal notable differences between the microbiota of buffalo and cow milk. Buffalo milk can inherently support probiotic bacteria more effectively than cow milk, likely due to its higher buffering capacity and nutrient density (Simões da Silva et al. 2020). Traditional fermented buffalo milk products (such as Indian dahi or Sri Lankan buffalo curd) harbor rich communities of wild LAB with broad probiotic properties (Abesinghe et al. 2020), reflecting buffalo milk's suitability for spontaneous fermentation. In direct comparisons, fermented buffalo milk beverages exhibit slower post‐acidification and higher survival rates of probiotics (e.g., L. acidophilus) during in vitro digestion when compared to equivalent cow milk products (Simões da Silva et al. 2020). These findings suggest that the buffalo milk matrix creates a more favorable environment for probiotic viability. At the same time, buffalo milk can present unique microbial challenges—for instance, higher indigenous yeast counts, which require careful quality control. Overall, such differences highlight the importance of tailoring fermentation practices to buffalo milk and underscore the need for further comparative research to fully leverage buffalo milk's microbial advantages while managing any spoilage risks.

Studies utilizing both culture‐dependent and independent methods, such as 16S rRNA sequencing, have revealed a rich and complex microbiota in buffalo milk (Table 3). The predominant bacterial genera include Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, and Enterococcus, which play crucial roles in beneficial fermentation processes (Vargas‐Ramella et al. 2021). Among LAB strains, such as Lactobacillus delbrueckii, L. acidophilus, and S. thermophilus, are particularly significant due to their contributions to fermentation, texture enhancement, and flavor development in dairy products. Furthermore, LAB offers natural bioprotection by producing organic acids, bacteriocins, and other antimicrobial compounds that inhibit spoilage and pathogenic bacteria. Compared to cow milk, buffalo milk may support higher abundance and survivability of certain LAB strains, like L. bulgaricus and E. faecium, due to its richer nutritional matrix and buffering capacity (Simões da Silva et al. 2020). Both milk types share dominant bacterial phyla (Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes), but they differ in species prevalence and function. For example, buffalo‐derived Lactococcus lactis shows higher exopolysaccharide (EPS) production (Guo et al. 2024; Vargas‐Ramella et al. 2021).

TABLE 3.

Probiotic and functional bacterial strains identified in buffalo milk: Research‐based insights.

Identified strain	Country of study	Identification method	Sample source	Probiotic properties and functional traits	Future research and applications	References
Lactiplantibacillus collinoides Lactobacillus sake and Lactobacillus alimentarius	Iran	16S rRNA gene sequencing	Cow milk, buffalo milk, cheese, and yoghurt	Antimicrobial activity against Staphylococcus aureus (ATCC‐6538), Bacillus subtilis (ATCC‐12711), and Pseudomonas aeruginosa (ATCC‐27853)	Potential use in food, pharmaceutical, and functional food production	Karami et al. (2017)
Lactobacillus plantarum Lactobacillus herbarum	Murthal, Sonepat, Haryana of India	16S rRNA gene sequencing	Cow milk, and buffalo milk	Antimicrobial activity against Escherichia coli, P. aeruginosa, Listeria monocytogenes, Bacillus cereus, S. aureus, and Shigella flexneri	A bacteriocin‐producing strain, It exhibits potential for novel bacteriocin production; further characterization is needed	Parveen and Nehra (2020)
Weissella confusa (NMCC‐M2), Leuconostoc pseudomesenteroides (NMCC‐M4), Lactococcus lactis subsp. hordniae (NMCC‐M5), Enterococcus faecium (NMCC‐M6), and Enterococcus. lactis (NMCC‐M7)	Islamabad, Pakistan	16S rRNA gene sequencing	Raw buffalo milk	Antimicrobial activity against Salmonella typhimurium (ATCC 14028), E. coli (ATCC 8739), S. aureus (ATCC 6538), and B. cereus (ATCC‐11778), acid and bile tolerance, enzymatic potential (proteolytic activity, lypolytic activity and amylolytic activity), cholesterol reduction, antibiotic susceptibility, and hemolytic activity	NMCC‐M2 strain holds promise for cholesterol‐lowering probiotic applications in fermented food products	Hameed et al. (2022)
L. lactis, Lacticaseibacillus paracasei Lactiplantibacillus paraplantarum Leu. spp. Enterococcus spp., Streptococcus spp., Vagococcus fluvialis, and Pediococcus pentosaceus from dairy products No specific strain from buffalo milk or cheese	South Korea	16S rRNA gene sequencing	Cow milk and cheese, Buffalo milk cheese, and fermented cow milk	Acid and bile tolerance, viability after simulated gastrointestinal passage, hydrophobicity, zeta potential, and mucin adhesion	Strains require metabolic activity assays, virulence and resistance gene screening, and animal studies before human applications	Arellano et al. (2024)
Weissella paramesenteroides (FT 351), L. lactis (FT 359), W. confusa (FT 424), Weissella hellenica (FT 476), Enterococcus faecalis (FT 522) identified from buffalo milk; L. lactis (FT 609), Enterococcus hirae (FT 655), L. mesenteroides (FT 664) and L. citreum (FT 671) from buffalo cheese	Brazil	16S rRNA gene sequencing	Cow, buffalo and goat milk and cheeses (from which milk?)	Antifungal activity against Penicillium expansum and Yarrowia lipolytica and proteolytic activity	Potential for dairy product enhancement, shelf‐life extension, and novel fermented product development	Tulini et al. (2016)
Lacticaseibacillus rhamnosus (LB1.5) and L. paracasei (LB6.4)	Brazil	Combination of MALDI‐TOF and partial 16S rDNA sequencing	Raw buffalo milk	Antimicrobial activity against E. coli, P. aeruginosa, L. monocytogenes, and S. aureus, milk proteolysis and exopolysaccharides (EPSs) production, adhesion to Caco‐2 cells, coaggregation with S. aureus and E. coli	Both strains identified as promising probiotic candidates for further in vivo studies	Breyer et al. (2020)
L. Lactis	India	16S rRNA gene sequencing	Cow, sheep, goat, camel and buffalo milk	Lactic acid production	Further in vitro and in vivo studies required	Sharma et al. (2013)
Limosilactobacillus fermentum strain L 23, L. fermentum strain 6704 and Lactobacillus oris strain J‐1	Indonesia	16S rRNA gene sequence analysis	Raw buffalo milk	Antimicrobial activity against L. monocytogenes	Further in vitro and in vivo studies required	Melia et al. (2017)
L. plantarum	West Sumatera, Indonesia	16S rRNA gene sequence analysis	Fermented Buffalo milk	Antimicrobial activity against E. coli, S. aureus, and S. typhi and acid tolerance (pH 3–4)	Further in vitro and in vivo studies required	Syukur et al. (2014)
L. plantarum, Lactobacillus pentosus, L. fermentum, and Limosilactobacillus reuteri	Thailand	16S rRNA gene sequence analysis	Raw buffalo milk	Acid, bile, and lysozyme resistance; tolerance to gastrointestinal fluids; adhesion ability; antibacterial activity; EPS production	Strains show promise as starter cultures for functional foods due to resilience against environmental stress and pathogen inhibition	Kalhoro (2019)
Four lactic acid bacteria isolates	Bangladesh	Culture‐based characterization	Buffalo milk	NaCl and phenol tolerance test, bile salt tolerance, and antimicrobial activity against Salmonella thyphimurium, E. coli, Shigella spp. and Vibrio cholerae	Further identification using 16S rRNA sequencing and molecular methods recommended	Forhad et al. (2016)

Table 3 summarizes key studies that have characterized the bacterial communities in buffalo milk, highlighting specific microbiota with potential probiotic applications. Many of these LAB strains, identified through 16S rRNA gene sequencing and other molecular techniques, exhibit promising probiotic properties, including antimicrobial activity, acid and bile tolerance, cholesterol‐lowering effects, and enzymatic capabilities (Arellano et al. 2024; Hameed et al. 2022; Kalhoro 2019). The microbial diversity of buffalo milk is also shaped by the sample source. Raw milk yields more diverse wild LAB strains suitable for starter cultures, whereas fermented dairy products favor robust strains with enhanced functional properties.

The identification of probiotic and functional bacterial strains in buffalo milk primarily relies on 16S rRNA gene sequencing, widely regarded for its accuracy and phylogenetic resolution. However, alternative methods, including MALDI‐TOF mass spectrometry combined with partial 16S rDNA sequencing, have been adopted in advanced research settings for faster and more precise identification (Breyer et al. 2020). Traditional culture‐based methods, though still in use, offer limited resolution and often require molecular validation for precise classification (Forhad et al. 2016). The microbial diversity of buffalo milk is also shaped by the sample source. Raw milk tends to yield a broader range of wild LAB strains suitable for starter cultures, whereas fermented dairy products favor robust strains with enhanced functional properties. Regional disparities in research infrastructure further influence identification approaches, with some developing regions relying on culture‐based techniques or 16S rRNA sequencing alone, whereas technologically advanced centers integrate MALDI‐TOF and sequencing combinations for greater accuracy. Future research should explore metagenomic sequencing and shotgun metagenomics to deepen insights into buffalo milk microbiota beyond the limitations of 16S rRNA‐based methods.

Table 3 also clearly reveals that microbial species of buffalo milk differ across countries, suggesting that environmental factors such as climate, temperature, humidity, and soil composition significantly influence their microbial composition and distribution. These regional variations underscore the importance of local research and probiotic characterization. Strain‐specific functionalities observed across geographies may inform targeted probiotic development tailored to population‐specific needs. Furthermore, regional studies indicate country‐specific variations in probiotic strain functionality. For instance, research in China highlights the vitamin B production capabilities of L. plantarum, whereas studies in Canada have shown its cholesterol‐reducing effects. Additionally, the functional performance of probiotic strains such as EPS production, biofilm formation, and matrix compatibility can be influenced by both the microbial origin and fermentation conditions, highlighting the importance of context‐specific strain selection and validation. Similarly, L. delbrueckii has demonstrated anti‐colitis and metabolic health benefits in studies conducted in Taiwan and India. These variations underscore the importance of localized research and suggest that buffalo milk microbiota may be uniquely adapted to their regional environments, aligning with their role in supporting sustainable food systems in areas such as South Asia and Latin America.

Table 4 provides a comprehensive overview of the potential health benefits of probiotic microorganisms isolated from buffalo milk. Initial research supports their probiotic potential, demonstrating antimicrobial activity, acid and bile tolerance, cholesterol‐lowering effects, and enzymatic capabilities. However, further in vivo studies are necessary to validate their efficacy in human health and explore their applications in food safety, biotechnology, and functional food development.

TABLE 4.

Probiotic potential and health benefits of microorganisms isolated from buffalo milk.

Genus	Species	Source/milk type	Technological functions	Documented potential health benefits	Application in dairy products	Country of study	Key country‐specific findings	References
Lactobacillus	Lactobacillus plantarum	Buffalo milk; dahi; fermented milk product	Improves safety and quality of dairy products; proteolysis, lipolysis, and aroma compounds	Produces bacteriocins and vitamin B; enhances vitamin and mineral absorption; stimulates organic acid and amino acid production; provides protection against pathogens through competitive exclusion; teichoic acid linked to anti‐inflammatory activity	Cheese; fermented milk (commercial); fermented soy milk; kefir; yoghurt; encapsulated bacteria; rose‐hip drink; commercial probiotic mixture	China; France; Canada; Brazil	Vitamin B production and organic acid generation observed in China	Barreto et al. (2014), Li and Gu (2016), Lee et al. (2017), Plaza‐Diaz et al. (2019), Vargas‐Ramella et al. (2021)
							Anti‐inflammatory activity highlighted in France and Canada
							Total cholesterol, low‐density lipoprotein cholesterol (LDL) and ‐glutamyl transpeptidase reduction reported in Brazil
	L. paracasei	Raw cow milk; fermented milk; cheese; buffalo milk		Anti‐obesity effects; improves immune function via NK cell activity; modulates short‐chain fatty acids and volatile compounds beneficial for diarrhea and constipation	Fermented milk; fermented soy milk; commercial probiotic mixture	Canada; Korea; India	Immune‐modulating properties studied in Korea	Lee et al. (2017), Rajkumar et al. (2014), Lee et al. (2017), Plaza‐Diaz et al. (2019); Vargas‐Ramella et al. (2021)
							Gut microbiome modulation reported in Canada
							Anti‐obesity effects observed in India and Korea
	Limosilactobacillus fermentum	Buffalo milk; fermented milk; dairy products	Proteolysis, lipolysis, and aroma compounds	High ferulic acid esterase activity improves bioavailability of ferulic acid when orally ingested in a microencapsulated form	Cheese; commercial probiotic mixture; microencapsulated probiotic	Canada; India; Australia	Ferulic acid bioavailability enhancement studied in Canada	Bhathena et al. (2009), Vargas‐Ramella et al. (2021)
							Total cholesterol, LDL cholesterol, and triglyceride reduction studied in Canada
							Probiotic activity in dairy fermentation focused in India
							Atopic dermatitis treatment observed in Perth, Western Australia
Lactobacillus delbrueckii		Buffalo milk; yoghurt; cheese		Binds iron hydroxide, reducing pathogen availability; colonizes gut, regulates microflora, withstands acidity, and inhibits pathogens	Cheese; commercial probiotic mixture	Taiwan; India	Overweight beneficial effects: HDL cholesterol, insulin sensitivity, and amelioration of inflammation (hsCRP) studied in India	Oelschlaeger (2010), Shiby and Mishra (2013), Rajkumar et al. (2014), Vargas‐Ramella et al. (2021)
							Anti‐colitis effect observed in Taiwan
	Lactobacillus kefiranofaciens	Buffalo milk; kefir grain	Proteolysis, lipolysis, and aroma compounds; highly stress‐tolerant, withstanding heat (52°C), cold (−20°C), acid (pH 3.0), and bile salts (0.2%)	Produces kefiran; inhibits biofilm formation; reduces effects of enterohemorrhagic Escherichia coli infection, bacterial translocation, and intestinal issues	Kefir grains	Egypt; Taiwan	Enterohemorrhagic E. coli (EHEC) preventing infection and its effects studied in Taiwan	Chen et al. (2012), Chen et al. (2017), Hong et al. (2010), Vargas‐Ramella et al. (2021)
							Antiallergic effect observed in Taiwan
Streptococcus	Streptococcus thermophilus	Buffalo milk cow milk; dahi; cheese	Starter culture, lactose to lactate; exopolysaccharide and bacterioncin production	Produces extracellular polysaccharides, bacteriocins; establishes intestinal microflora, protects gut health; generates folate for DNA repair; regulates immune response, reducing inflammation	Yoghurt; Cheese; commercial probiotic mixture; tablet with Lactobacillus bulgaricus	France; Taiwan; India; Spain	Child obesity and nonalcoholic fatty liver disease (steatohepatitis) effects in Italy and Spain	Iyer et al. (2010), Oelschlaeger (2010), Aller et al. (2011), Shiby and Mishra (2013), Alisi et al. (2014), Plaza‐Diaz et al. (2019), Vargas‐Ramella et al. (2021)
	Streptococcus macedonicus	Buffalo milk; fermented Greek kasseri cheese from sheep milk		Similar to S. thermophilus with no pathogenicity; some strains produce macedocin, a food‐grade bacteriocin; does not survive stomach acidity	Yoghurt, cheese; commercial probiotic mixture	Northern Greece; Belgium; Italy	Overweight beneficial effects: HDL cholesterol, insulin sensitivity and amelioration of inflammation (hsCRP) observed in India	De Vuyst and Tsakalidou (2008), Rajkumar et al. (2014), Vargas‐Ramella et al. (2021)
Lactococcus	Lactococcus lactis	Buffalo milk; kefir; yoghurt, koumiss and goat yoghurt	Key industrial LAB; starter cultures; flavor compounds; acidification; proteolysis; citrate utilization; fat metabolism; bacteriocin production	Fermented milk provides cardiovascular benefits, reducing blood pressure, LDL cholesterol, and triglycerides; survives low pH and bile salts; co‐aggregates with E. coli, potential probiotic starter	Fermented milk	Mexico; the Netherlands; Denmark	Blood pressure lowering effect studied in Mexico	Iyer et al. (2010), Sabir et al. (2010), Beltrán‐Barrientos et al. (2018), Vargas‐Ramella et al. (2021), Yu et al. (2024)
							Atopic dermatitis (eczema) prevention effects found in the Netherlands
							Overweight treatment: LDL cholesterol reduction and fibrinogen increase studied in Denmark
Leuconostoc	L. lactis	Buffalo milk; camel milk; pasteurized milk	CO2 production; lactose and citrate metabolization; bacteriocin production	Produces bioactive peptides with antioxidant and ACE‐inhibitory activity; potential use in nutraceuticals and functional foods	Fermented milk	Iran	Blood pressure regulation (antioxidant and ACE‐I activities) observed in Iran	D'Angelo et al. (2017), Soleymanzadeh et al. (2019), Vargas‐Ramella et al. (2021)
Pseudomonas	Pseudomonas fragi	Raw buffalo milk; cheese; raw cow milk	Produces fruity off‐odors in dairy products due to methyl esters and short‐chain ethyl esters	Spoilage of pasteurized and UHT milk via thermostable proteases and lipases; generates volatile compounds like methyl and ethyl acetate, ketones, alcohols, and sulfur compounds	Milk and its products	Spain; Australia	—	Morales et al. (2005), Stanborough et al. (2018), Vargas‐Ramella et al. (2021)

Although LAB remain the primary focus due to their well‐documented beneficial roles, buffalo milk also harbors non‐LAB microorganisms, including Pseudomonas, Enterobacter, Bacillus, and Staphylococcus species (Vargas‐Ramella et al. 2021). These bacteria can pose challenges to product stability, quality, and safety by contributing to spoilage and contamination. However, certain non‐LAB species may positively influence specific fermentation environments, enhancing microbial interactions that improve the flavor, texture, and shelf life of dairy products. Further research is required to elucidate their precise roles and potential benefits in dairy applications.

Studies have also highlighted the technological functionalities of LAB in dairy processing. Many strains from buffalo milk, including L. plantarum, L. fermentum, L. delbrueckii, and Lactococcus lactis, exhibit proteolytic, lipolytic, and EPS producing abilities, which contribute to improved dairy product quality and sensory properties. Additionally, certain probiotic strains demonstrate anti‐inflammatory, cholesterol‐lowering, and gut health‐enhancing effects, making them promising candidates for incorporation into functional dairy foods (Arellano et al. 2024; Hameed et al. 2022; Kalhoro 2019).

Given the promising health benefits and technological potential of probiotic strains isolated from buffalo milk, future research should prioritize in vivo studies, metabolic activity assays, and clinical evaluations. Expanding identification techniques, including metagenomic sequencing and shotgun metagenomics, will facilitate the discovery of novel probiotic strains and support the development of buffalo milk‐based functional dairy products with enhanced health benefits and technological applications.

4. Development of Buffalo Milk Probiotic Dairy Products

Enhancing the value of buffalo milk and its derivative products is key to improving the competitiveness and sustainability of the buffalo milk industry. Although buffalo milk has long been valued for its nutritional benefits and cultural significance, particularly in South Asia, shifting dietary patterns and global population growth are driving increased demand for dairy products. The unique microbiota of buffalo milk offers significant advantages for the production of fermented dairy products, particularly yoghurt and mozzarella cheese. Its higher protein and fat content, coupled with its distinct microbial community, enhances the creaminess, texture, and depth of flavor compared to cow milk‐based products (Emakpor et al. 2024).

The utilization of LAB strains naturally present in buffalo milk as starter cultures can significantly improve product quality, microbial stability, and safety. These strains can be selected on the basis of their probiotic potential, technological properties (e.g., acidification speed, proteolytic, and lipolytic activity), and their role in extending shelf life. The isolation of unique probiotic cultures from buffalo milk represents an opportunity for functional food development, aligning with increasing consumer demand for health‐promoting dairy products. Additionally, buffalo milk can serve as an alternative dairy base, benefiting from value‐added product innovation, including the development of cheese, ghee, mozzarella, and protein powders, with enhanced quality and extended shelf life through innovative food processing techniques.

4.1. New Probiotic Strains From Buffalo Milk

LAB are the dominant bacterial group in buffalo milk and play a crucial role in the production of fermented dairy products like yoghurt, cheese, and kefir. Their metabolic activity, which involves converting lactose to lactic acid, lowers the milk's pH leading to coagulation and texture development in fermented products (Coelho et al. 2022). LAB also contributes to the formation of volatile compounds, influencing the sensory characteristics and overall flavor profile of dairy products. The probiotic potential of LAB strains from buffalo milk is increasingly being investigated. Specific strains, such as L. plantarum and Lacticaseibacillus casei, have demonstrated the ability to survive gastrointestinal conditions, adhere to intestinal cells, and inhibit pathogenic microorganisms such as Clostridium difficile (Agolino et al. 2024). These probiotic strains are associated with various health benefits, including improved gut microbiota balance, enhanced immune function, and alleviation of lactose intolerance symptoms. Additionally, buffalo milk‐derived probiotic cultures can synthesize other functional metabolites. For example, certain Lactobacillus and Pediococcus isolates from buffalo milk have demonstrated robust conjugated linoleic acid (CLA) production during fermentation (Rekowsky et al. 2025), enhancing the nutraceutical profile of buffalo milk‐based fermented products. Moreover, LAB isolated from traditional buffalo curds such as dadih have shown notable antimicrobial activity, suggesting the presence of bacteriocin‐producing strains with promising applications for natural food preservation (Ginting et al. 2025). A review by Vargas‐Ramella et al. (2021) confirmed that raw buffalo milk contains a diverse range of probiotic microorganisms that can be effectively utilized in the high‐quality traditional dairy production, enhancing both product value and health benefits. Figure 2 presents an overview of probiotic bacterial genera isolated from buffalo milk, including their functional properties, associated health benefits, and potential implications for future research and applications in functional dairy products.

FIGURE 2.

Functional and health potential of buffalo milk‐derived probiotics: Research insights.

4.1.1. Selection Criteria

The selection of novel probiotic strains for dairy products is a critical process that requires careful evaluation to ensure the efficacy, safety, health benefits, and commercial viability of the final product. Key selection criteria include:

• Health benefits: The chosen probiotic strain must be supported by robust scientific evidence suggesting health benefits, such as improved digestive health, enhanced immune function, or other therapeutic effects (Ma et al. 2023). For example, Limosilactobacillus reuteri and Bifidobacterium longum are selected for their potential to reduce inflammation and strengthen gut barrier function (Hoy‐Schulz et al. 2015). The richer fat content and bioactive compounds of buffalo milk may provide extra health benefits, including improved gut health, stronger immune responses, and better nutrient absorption (Vargas‐Ramella et al. 2021). The documented health benefits of these strains are summarized in Table 4.

• Survivability and stability: The strain must remain viable throughout the entire production process, including fermentation, drying, and storage, while maintaining its effectiveness over the product's shelf life. It must also withstand the acidic environment of the stomach to reach the intestine in sufficient quantities (Fenster et al. 2019).

• Compatibility with dairy matrix: The strain should complement the dairy matrix, ensuring that it does not negatively impact taste, texture, or aroma. Ideally, the probiotic strain should also enhance the fermentation process, contributing to better flavor and texture (de Souza et al. 2024).

• Safety and regulatory approval: The selected strains must be safe for human consumption, free from pathogenic effects, and supported by clinical trials that meet scientific standards. However, obtaining robust clinical validation for a novel strain remains a significant challenge. High costs, extended trial durations, and the need for large sample sizes to confirm therapeutic efficacy can hinder regulatory approval. These barriers are particularly relevant for probiotic development in less‐studied matrices like buffalo milk, where fewer precedents exist to streamline regulatory pathways.

The integration of probiotic strains derived from buffalo milk into functional dairy products has the potential to revolutionize the industry, providing nutritionally enhanced alternatives while promoting sustainability and biodiversity in dairy production. Future research should focus on optimizing probiotic fermentation processes, validating health claims through clinical studies, and expanding product development strategies to meet growing consumer demand for innovative and health‐focused dairy products. Additionally, expanding the utilization of buffalo milk could also support sustainable livestock practices by promoting genetic diversity and fostering the evaluation of different buffalo breeds for food production.

4.2. Processing Techniques for Buffalo Milk‐Based Products

The production of dairy products involves both conventional and innovative techniques, each playing a crucial role in ensuring product quality, safety, and functionality. Figure 3 provides an overview of these methods, with emphasis on their specific relevance to buffalo milk's composition and the development of probiotic products. Due to its larger fat globules and higher fat and protein content, buffalo milk presents distinct challenges and opportunities in processing. Homogenization is critical to reduce fat globule size, improve emulsion stability, and enhance texture, especially in products such as yoghurt and fermented milk. Nano‐homogenization techniques are being explored to optimize fat dispersion in buffalo milk, where conventional homogenization may be less efficient (Aliabbasi and Emam‐Djomeh 2024; Mejares et al. 2022).

FIGURE 3.

Techniques for production of dairy products.

Maintaining probiotic viability during processing and storage is a central challenge in developing functional dairy products. Innovative techniques such as microencapsulation, electrospinning, ultrasound, and high‐pressure processing (HPP) have been applied or adapted for buffalo milk to address this need.

Microencapsulation enhances probiotic survival by protecting microorganisms during food processing, storage, and gastrointestinal transit. Various encapsulation materials, including alginate, chitosan, milk proteins, and carbohydrates, have been employed to create stable probiotic formulations. In buffalo milk, Frakolaki et al. (2021) showed that encapsulating L. acidophilus with sodium alginate and whey protein isolates improved survival during gastrointestinal simulation. The higher fat and protein content of buffalo milk contributes to greater stability of encapsulated probiotics.

Electrospinning, a nanotechnology‐based encapsulation method, forms ultrafine fibers that protect probiotics from environmental stressors and allow for controlled release. Similarly, hydrogel encapsulation maintains hydration around probiotics, improving their viability and functional performance in products such as probiotic cheese coatings (Sun et al. 2023). These technologies offer benefits for shelf life, delivery, and sensory properties but remain underutilized in industrial‐scale buffalo milk applications. In parallel with advances in encapsulation and fermentation optimization, post‐fermentation drying methods are being refined for buffalo milk‐based probiotic foods. Notably, freeze‐drying enables the conversion of cultured buffalo milk into shelf‐stable probiotic powders, extending product shelf life while preserving microbial viability for broader distribution.

HPP is a non‐thermal preservation method that enhances microbial safety and maintains nutritional integrity. In buffalo milk, HPP induces structural changes in proteins, such as casein micelle enlargement and whey protein denaturation, which can influence texture and digestibility (Huppertz et al. 2005). HPP‐treated buffalo cheese has shown improved textural properties and safety while preserving probiotic functionality (Falih et al. 2024).

Ultrasound is another non‐thermal technology used to improve probiotic delivery in buffalo milk. It enhances fermentation by promoting LAB metabolic activity and accelerating acidification. At 20 kHz, ultrasound serves as an effective alternative to high‐shear homogenization, reducing fat globule size and improving product uniformity and texture. Abesinghe et al. (2022) and Pacheco et al. (2023) both demonstrated that ultrasound treatment of buffalo milk enhanced fermentation efficiency, improved rheological properties, and increased the survival of probiotic cultures.

Advanced fermentation technologies allow for precise control of temperature, pH, and fermentation time‐critical factors for consistent quality in buffalo milk products. Buffalo milk's higher buffering capacity requires close pH regulation to ensure optimal growth conditions for probiotic strains (Mendonça et al. 2022; Siddiqui et al. 2023).

Finally, synbiotic formulations that combine probiotics with prebiotics such as inulin or fructooligosaccharides (FOS) enhance gut health benefits and product stability. These prebiotics selectively promote LAB activity, supporting microbial balance and extending shelf life (Pandey et al. 2015). As shown in Table 5, conventional techniques remain fundamental to dairy processing, but innovative technologies offer opportunities to enhance probiotic viability, functional properties, and sensory attributes. Collectively, these emerging technologies present valuable opportunities to enhance the viability, delivery, and efficacy of probiotics in buffalo milk‐based products. Future research should focus on optimizing microencapsulation strategies, expanding the industrial application of HPP and ultrasound, and developing synbiotic systems tailored to buffalo milk matrices. Figure 3 summarizes these conventional and innovative techniques and their applications in buffalo milk‐based product development.

TABLE 5.

Techniques for production of dairy products.

Technology		Description	Applications in dairy products	Benefits	Challenges and future directions	References
Conventional	Standardization	Adjusting the composition to achieve consistent quality, texture, and nutritional value. This involves regulating fat content, protein levels, and total solids to meet industry and regulatory standards	Milk, cheese, yoghurt, fermented dairy products, butter, cream, milk powders	Ensures product consistency and quality, improves processing efficiency and yield in dairy manufacturing, helps to meet nutritional and regulatory requirements thus enhances consumer trust and marketability	Variations in raw milk composition, balancing between nutrition, taste, and regulatory compliance are complex. Future advancements in AI and automation	Mcsweeney and Fox (2013), Sfakianakis and Tzia (2014)
	Homogenization	Mechanical process that breaks down fat globules in milk to create a uniform and stable emulsion. This prevents cream separation, improves texture, and enhances the sensory quality of dairy products	Milk, yoghurt, fermented products, ice cream, cheese, cream, and butter	Enhances texture, stability, and shelf life, improves nutrient absorption and digestibility, making dairy products more consumer‐friendly. The process also enhances flavor and appearance, contributing to better market acceptance	Energy‐intensive and costly, homogenization may affect flavor through oxidation and risk nutrient loss. Future advancements in nano‐homogenization may improve stability and efficiency	Aliabbasi and Emam‐Djomeh (2024)
	Pasteurization	A heat treatment process (e.g., 72°C for 15 s for HTST) designed to kill pathogenic microorganisms while preserving most of the milk's nutritional and sensory qualities	Milk, cream, yoghurt, cheese, and kefir	Preserves flavor, texture, and most nutrients while ensuring safety, reduces the risk of foodborne illnesses	May not eliminate all spoilage organisms, shortening shelf life	Mcsweeney and Fox (2013), Sfakianakis and Tzia (2014)
	Sterilization	A more intense heat treatment (e.g., UHT at 135–150°C for 2–5 s or in‐container sterilization at 110–120°C for 20–30 min)	UHT milk, evaporated milk, and canned dairy products	Extends shelf life (up to several months) without refrigeration, reduces the risk of foodborne illnesses	Can alter flavor, texture, and nutrient content. Innovations are being explored to enhance microbial safety while preserving the quality of dairy products	Mcsweeney and Fox (2013), Sfakianakis and Tzia (2014)
	Traditional fermentation	Use of natural or selected microorganisms (lactic acid bacteria, yeasts, molds) to convert lactose into lactic acid	Yoghurt, kefir, cheese, buttermilk, and lassi	Rich in probiotics, improve shelf life, nutritional content, flavor and texture	Inconsistent product quality due to variability in microbial strains, the need for proper storage conditions to maintain microbial viability, and consumer hesitance towards adopting fermented dairy products Future research focusing on optimizing microbial strains and exploring innovative fermentation technologies	Sfakianakis and Tzia (2014), Gänzle et al. (2023)
	Spray drying	Sprayed into a hot air chamber, causing rapid evaporation and forming fine powder	Powdered milk, whey powder, infant formula, and milk‐based beverages	Rapid process, extends shelf life, easy to manage the fine powders	Loss of heat‐sensitive nutrients (e.g., vitamins) and flavor changes due to high temperatures	Mujumdar (2014)
	Freeze drying	Involves freezing the milk or dairy product, followed by a vacuum process that removes the water through sublimation	Long‐life dairy products (Freeze‐dried yoghurt, ice cream, and cheese powders), specialized dairy ingredients for culinary and pharmaceutical uses	Preserves the nutritional and sensory qualities of the original product, making it ideal for long‐term storage	High energy consumption and longer processing time, leading to higher costs. Technological advancements are needed to reduced energy consumption	Waghmare et al. (2024)
Innovative	Microencapsulation	Encapsulation of probiotic cells in biopolymeric coatings (e.g., alginate, chitosan, whey proteins)	Yoghurt, cheese, probiotic drinks, buffalo milk‐based products	Enhances probiotic survival during storage and digestion, improves sensory properties	Optimization required for different dairy matrices, cost implications	Iravani et al. (2014), Kowalska et al. (2022)
	Electrospinning and Hydrogel Encapsulation	Advanced encapsulation techniques improving probiotic delivery	Dairy‐based supplements, probiotic cheese coatings	Enhances probiotic protection and targeted release in the gut	Requires further studies for industrial‐scale dairy applications	Sun et al. (2023)
	HPP	Non‐thermal technology that inactivates spoilage microbes while preserving probiotics	Fermented dairy, buffalo milk yoghurt, kefir, and probiotic cheese	Retains probiotic viability, enhances texture and flavor, and improves safety	More research needed on its effects on different probiotic strains	EFSA Panel on Biological Hazards (BIOHAZ Panel) (2022); Falih et al. (2024)
	Ultrasound	Non‐thermal technology that uses high‐frequency sound waves (above 20 kHz) to enhance the quality, functionality, and safety of probiotic dairy products	Yoghurt; cheese; probiotic milk; beverages, ice cream, and plant‐based dairy	Improving microbial viability, optimizing fermentation, and enhancing texture and functionality	Further research on large‐scale applications and regulatory aspects for maximizing its commercial potential in the probiotic dairy industry	Abesinghe et al. (2020), Abesinghe et al. (2022), Akdeniz and Akalın (2022)
	Controlled fermentation	Precision control over temperature, pH, and fermentation time for probiotic growth	Yoghurt, kefir, traditional dairy cultures	Improves consistency, enhances probiotic stability and bioactivity	Requires advanced monitoring systems for large‐scale production	Siddiqui et al. (2023)
	Synbiotic formulation	Combining probiotics with prebiotics (e.g., inulin, fructooligosaccharides) to enhance probiotic effectiveness	Functional dairy, buffalo milk yoghurt, health drinks	Enhances probiotic survival, boosts gut microbiota diversity, improves health benefits	Selection of compatible probiotic‐prebiotic combinations required	Pandey et al. (2015)

4.3. Safety and Risk Assessment of Probiotic Buffalo Milk Products

Ensuring the safety and efficacy of probiotic dairy products is essential for consumer protection and regulatory compliance. Although global regulatory frameworks apply broadly across dairy products, buffalo milk‐based probiotic products must also comply with these standards, particularly when novel strains or formulations are involved.

Probiotic strains, whether derived from buffalo milk or not, must undergo rigorous assessment to confirm their safety, stability, and functionality. This includes evaluation of strain identity, antibiotic resistance, metabolic activity, and potential pathogenicity. International bodies such as the Codex Alimentarius Commission, the European Food Safety Authority (EFSA), and Food Standards Australia New Zealand (FSANZ) require scientific validation and safety documentation before health claims can be made (Table 6). Buffalo milk probiotic products are no exception, if a novel probiotic strain is isolated from buffalo milk, it would require GRAS notification in the United States or novel food approval under EFSA guidelines. In India, where buffalo milk is widely consumed, national guidelines for dairy probiotics apply uniformly, with no regulatory exemptions for buffalo‐derived strains.

TABLE 6.

Overview of probiotic regulatory standards worldwide.

Region	Regulatory body	Approval process	Health claim requirements	Examples of regulations	References
European union	European Food Safety Authority (EFSA)	Requires premarket approval and clinical validation of probiotic strains	Strict requirements for scientific substantiation before claims can be made	EFSA QPS (Qualified Presumption of Safety), EU Regulation (EC) No 1924/2006	EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) (2009)
United States	Food and Drug Administration (FDA)	Probiotics classified as dietary supplements or functional foods, requiring GRAS status	Claims must be substantiated but are generally less restrictive than EFSA	DSHEA (Dietary Supplement Health and Education Act)	US Food Drug Administration (2009)
Japan	Consumer Affairs Agency	Probiotics regulated under FOSHU (Foods for Specified Health Use)	Requires evidence‐based health claims approval process	FOSHU regulatory system	Shimizu (2003)
South Korea	Ministry of Food and Drug Safety (MFDS)	Regulated under Health Functional Foods (HFF)	Requires scientific validation and pre‐market approval for functional claims	Functional Health Foods Act	MFDS (2021)
China	National Health Commission (NHC)	Probiotics are classified under Food for Special Medical Purposes (FSMP)	Requires submission of safety and efficacy data for health claim approval	GB 7718‐2011 National Food Safety Standards	NHC (2021)
India	Food Safety and Standards Authority of India (FSSAI)	Regulated under the Food Safety and Standards Act	Requires scientific substantiation for claims, approval needed for novel probiotics	FSSAI Guidelines on Probiotic Foods	Food Safety and Standards Authority of India (FSSAI) (2021)
Australia and New Zealand	FSANZ (Food Standards Australia New Zealand)	Requires probiotics to be GRAS or classified as a novel food	Claims must be scientifically validated and follow food standard regulations	Standard 1.2.7: Nutrition, Health, and Related Claims	Food Standards Australia New Zealand (FSANZ) (2024)
Brazil and Argentina	ANVISA (Brazil) and National Food Institute (Argentina)	Adapted from Codex standards; probiotic approval varies by product	Certain health claims require clinical evidence; others follow Codex Alimentarius guidelines	ANVISA RDC 241/2018 (Brazil)	Brazil's National Health Surveillance Agency (ANVISA) (2018), 2023 Codex Alimentarius (2021)

Raw buffalo milk may harbor microbial hazards similar to those found in cow milk, including E. coli, Listeria monocytogenes, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. In regions where Brucella spp. is endemic, brucellosis remains a concern associated with unpasteurized buffalo milk, and rare cases of buffalopox virus have also been reported (Khurana et al. 2021; Kalhoro et al. 2023). These pathogens pose significant health risks if proper thermal processing is not implemented. Though no major fatalities from buffalo milk‐derived probiotic products have been reported, the potential risks highlight the critical need for pasteurization and rigorous hygiene in buffalo milk processing.

The probiotic safety assessment in buffalo milk matrices follows general standards but must also consider local microbial contexts. Studies such as Hameed et al. (2022) have tested buffalo milk‐derived LAB strains for antibiotic resistance and hemolytic activity, confirming their suitability for human use. Key safety parameters include the absence of toxic metabolite production, non‐hemolytic behavior, and lack of transferable antibiotic resistance genes. Modern safety evaluations also increasingly incorporate genomic tools. For instance, whole‐genome sequencing (WGS) of candidate strains enables detection of virulence factors, antibiotic resistance genes, and other potential risks, ensuring that only genetically safe organisms are considered for commercial use (Arellano et al. 2024). Any novel LAB strain derived from buffalo milk typically undergoes a stringent validation pipeline, beginning with in vitro assays to assess antibiotic susceptibility and toxin production, followed by animal studies for preliminary safety, and ultimately culminating in human clinical trials to confirm safety and efficacy. Maintaining this thorough vetting process is crucial for building regulatory and consumer confidence in buffalo milk probiotic products.

Buffalo milk itself plays a functional role in enhancing probiotic viability. Compared to cow milk, buffalo milk's higher fat content and buffering capacity create a more favorable environment for probiotic survival (Yuliana et al. 2023). Products such as buffalo milk yoghurt and cheese have demonstrated improved stability of probiotic populations, owing to slower pH decline during fermentation and reduced oxidative stress. Maintaining a therapeutic threshold of at least 10⁶ CFU/g requires optimized formulations and cold‐chain storage conditions.

Although in vitro and animal studies have demonstrated promising probiotic properties of strains derived from buffalo milk, future clinical trials are essential to validate their safety, efficacy, and health benefits in humans (Maftei et al. 2024). Such validation will support regulatory approval and guide their formulation into effective, evidence‐based functional foods (Brownie et al. 2015; Adejumo et al. 2023; Fijan 2014; Singh et al. 2024).

In summary, although the safety framework for probiotic dairy products is shared across milk types, buffalo milk brings unique considerations both in risk and opportunity. Microbial hazards must be carefully managed through pasteurization and hygiene, whereas probiotic strains—whether novel or established—must meet stringent global safety criteria. Furthermore, buffalo milk's physicochemical characteristics can positively influence probiotic stability and delivery, making it a promising medium for developing safe and effective functional dairy products.

4.4. High Throughput Microbial Analysis of Probiotic Dairy Products

Advancements in microbial analysis techniques have significantly improved the ability to assess the safety, efficacy, and composition of probiotic dairy products. These high‐throughput techniques provide detailed insights into microbial diversity, genetic characteristics, and probiotic functionality.

Metagenomics is a powerful approach that allows for the comprehensive identification of microbial communities without the need for cultivation. This technique enables researchers to analyze the abundance and functional roles of probiotic strains and detect genes associated with beneficial or harmful traits, such as antibiotic resistance genes (Billington et al. 2021). Studies by Catozzi et al. (2020) and Salman et al. (2023) demonstrated the utility of metagenomics in identifying bacterial populations in buffalo milk, distinguishing variations in microbial diversity across different health conditions such as mastitis or environmental influences. Additionally, Li et al. (2024) used metagenomics to assess how different milking methods impact the bacterial diversity of raw buffalo milk, providing valuable insights for dairy safety and probiotic selection.

WGS offers a detailed genetic profile of probiotic strains, identifying genes linked to virulence factors, antibiotic resistance, and metabolic activities. WGS enhances strain authentication and ensures that probiotic dairy products contain correctly identified microorganisms with beneficial properties. Margalho et al. (2021) used WGS to screen wild LAB from Brazilian artisanal cheeses, identifying strains with biopreservation potential for dairy fermentation. The application of WGS in buffalo milk probiotic research could further validate strain safety, optimize functional properties, and improve product labeling accuracy.

4.5. Metabolomics Approach to Assess Nutritional Quality and Safety of Probiotic Dairy Products

Metabolomics is an emerging tool that provides a comprehensive analysis of metabolites produced during fermentation and digestion, offering insights into the nutritional quality, safety, and functional properties of probiotic dairy products.

From a nutritional perspective, metabolomics allows for the detection of bioactive compounds such as short‐chain fatty acids (SCFAs), peptides, and vitamins produced by probiotics during dairy fermentation. These compounds contribute to gut health, immune modulation, and metabolic regulation, enhancing the overall health benefits of probiotic dairy products (Saulnier et al. 2011).

Safety assessment through metabolomics focuses on identifying harmful metabolites, such as biogenic amines, which can pose risks if accumulated in high concentrations. By profiling the metabolic outputs of probiotic strains, researchers can monitor the formation of desirable and undesirable compounds, ensuring that probiotic dairy products remain safe for consumption (Gänzle et al. 2023).

Despite its growing application in dairy science, metabolomic studies specifically targeting buffalo milk‐based probiotics remain limited (Vargas‐Ramella et al. 2021). Future research in this area could provide new insights into the metabolic signatures unique to buffalo milk, leading to the development of higher‐quality, safer, and more functional probiotic dairy formulations. By integrating metabolomics with genomic and microbial analysis techniques, dairy manufacturers can further enhance product innovation and market competitiveness.

5. Future Directions in Buffalo Milk Probiotic Research

The development of novel probiotic dairy products presents a significant and transformative opportunity to advance public health and drive innovation within the functional food industry. Rising consumer demand for functional foods is fueling the rapid growth of probiotic dairy products, offering immense potential for nutritional innovation and health benefits. Despite advancements in production technologies, strain selection, and quality control, significant challenges remain in ensuring probiotic stability, optimizing formulations, and navigating diverse international regulatory frameworks.

Cutting‐edge analytical techniques such as metagenomics and metabolomics have emerged as essential tools for evaluating probiotic safety, efficacy, and metabolic activity, ensuring that probiotic dairy products meet the highest standards of scientific validation and consumer trust. However, research gaps persist in understanding the probiotic potential of buffalo milk, particularly regarding its unique microbial composition, technological applications, and health benefits. Expanding research into buffalo milk probiotics is crucial for unlocking new functional food innovations and addressing growing consumer interest in alternative dairy sources. Future clinical validation of probiotic strains derived from buffalo milk is essential to confirm their safety and efficacy in humans and to support their regulatory approval and integration into functional foods.

In the coming years, emerging probiotic strategies and sustainability considerations are expected to shape buffalo milk research and its applications. Multi‐strain probiotic formulations and personalized nutrition approaches could be explored to maximize synergistic effects and target specific health outcomes for consumers. The concept of postbiotics beneficial non‐viable microbial preparations also presents an intriguing avenue for buffalo milk products, potentially offering health benefits without the viability constraints of live cultures. Recent consensus recommendations define postbiotics as “preparations of inanimate microorganisms and/or their components that confer a health benefit on the host” (Salminen et al. 2021), expanding opportunities to use heat‐killed or otherwise inactivated buffalo‐derived microbes for safety‐sensitive populations. Moreover, advances in biotechnology offer the potential to genetically enhance probiotic strains, for example, by improving their stress tolerance or boosting the production of functional metabolites. However, such approaches must be pursued cautiously due to ethical considerations and complex regulatory requirements. Alongside these innovations, ensuring the environmental and socio‐economic sustainability of buffalo milk probiotic production is paramount. Buffaloes can be integrated into sustainable agriculture systems by leveraging their ability to thrive on low‐quality forages and in harsh climates, converting resources less suitable for cows into nutrient‐rich milk (FAO 2023). By expanding buffalo milk utilization, the dairy sector could reduce pressure on conventional cattle farming, promote agro‐biodiversity, and support rural livelihoods in regions where buffalo rearing is traditional (Hernandez and Bonilla‐Landaverry 2025). Aligning technological innovation with sustainability principles will be key to positioning buffalo milk as both a viable and sustainable component of the global probiotic dairy industry.

Importantly, the global buffalo milk supply continues to expand at approximately 2.5% per year‐a faster rate than cow milk, which could fuel further functional product development (Vargas‐Ramella et al. 2021). In this context, closer collaboration between researchers, industry stakeholders, and local dairy cooperatives could expedite the translation of buffalo probiotic innovations into practice. Such partnerships facilitate technology transfer and ensure that the benefits of buffalo milk functional foods reach smallholder producers and regional economies, aligning innovation with inclusive growth. Equally vital is raising consumer awareness. Targeted education campaigns and sensory marketing can help overcome hesitancy toward buffalo‐based products in emerging markets, supporting broader acceptance and demand. Future research should also prioritize EPS‐producing strains and regionally important milk sources such as buffalo milk, which offer unique compositional advantages and potential for probiotic innovation across diverse product platforms. In parallel, researchers should explore consumer acceptability and market perception of buffalo milk probiotic products in both traditional and non‐traditional markets. Sensory studies that incorporate cultural preferences and regional dietary patterns can help guide product formulation and marketing strategies. Additionally, life cycle assessments and sustainability evaluations of buffalo‐based probiotic foods can provide evidence of their role in environmentally conscious food systems. Understanding these social and environmental dimensions will be key to supporting policy development, investment, and widespread adoption of buffalo milk innovations at scale.

In summary, strategic investments in scientific research, regulatory alignment, and technological innovation will be key to positioning buffalo milk as a viable and sustainable component of the global probiotic dairy industry. With strong regulatory frameworks across multiple regions, continued scientific advancements, and a growing emphasis on functional health benefits, buffalo milk‐based probiotics have the potential to become an integral part of the evolving functional dairy market, offering nutritionally rich, microbiologically stable, and globally competitive probiotic products.

Author Contributions

Mst. Umme Habiba: conceptualization, investigation, writing – original draft, writing – review and editing, visualization, methodology, formal analysis, data curation. Mary Ann Augustin: investigation, writing – original draft, writing – review and editing. Cristian Varela: writing – review and editing. Helen Morris: writing – review and editing. Md. Morshedur Rahman: writing – review and editing. Hayriye Bozkurt: conceptualization, writing – original draft, writing – review and editing, project administration, supervision, funding acquisition, methodology.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

All data are presented in the current study.