Bovine milk microbiota: Key players, origins, and potential contributions to early-life gut development

Graphical abstract

Highlights

• •Dominant and core microorganisms in bovine milk contribute to the mammary health and milk quality.
• •Bovine milk microorganisms originate from the mammary gland via entero- and rumen-mammary pathway.
• •Resident microorganisms in mammary glands may be an initial source of bovine milk microorganisms.
• •Bovine milk microbiota supports early-life gut development by improving the intestinal microbiota and immune functions.

Abstract

Background

Bovine milk is a significant substitute for human breast milk and holds great importance in infant nutrition and health. Apart from essential nutrients, bovine milk also contains bioactive compounds, including a microbiota derived from milk itself rather than external sources of contamination.

Aim of review

Recognizing the profound impact of bovine milk microorganisms on future generations, our review focuses on exploring their composition, origins, functions, and applications.

Key scientific concepts of review

Some of the primary microorganisms found in bovine milk are also present in human milk. These microorganisms are likely transferred to the mammary gland through two pathways: the entero-mammary pathway and the rumen-mammary pathway. We also elucidated potential mechanisms by which milk microbiota contribute to infant intestinal development. The mechanisms include the enhancing of the intestinal microecological niche, promoting the maturation of immune system, strengthening the intestinal epithelial barrier function, and interacting with milk components (e.g., oligosaccharides) via cross-feeding effect. However, given the limited understanding of bovine milk microbiota, further studies are necessary to validate hypotheses regarding their origins and to explore their functions and potential applications in early intestinal development.

Introduction

Breast milk is often referred to as the “white blood” from the mother due to its rich nutritional composition, including essential nutrients such as proteins, fats, amino acids, vitamins, and more, as well as biologically active compounds like immunoglobulins, oligosaccharides, and growth factors [1]. The significance of breast milk microorganisms has garnered considerable attention due to their relevance to both the well-being of the offspring and the mother, alongside advancements in microbial detection techniques witnessed in recent decades [2], [3]. Bovine milk, the most available commercial milk, is produced by dairy cows and processed on a global scale. Although it cannot completely replace human milk, bovine milk can be used as a complementary food for infants during lactation to support growth and development [4]. The main advantage of using bovine milk is that it can be processed in batches and stored over time, overcoming the limitations of the cow's lactation cycle and providing a continuous supply of milk to humans of all ages. Bovine milk provides an ideal environment for the growth of microorganisms due to its suitable pH, temperature, and rich nutrients (Table 1). These microorganisms are acquired from external contamination as well as internal tissues and body fluids [5], [6]. During the past decade, research on the bovine milk microbiota has taken different directions. Conventional research has focused mainly on the effects of bovine milk microbes, especially external bacterial contaminants that may have adverse effects on mammary gland health and the quality of dairy products [7], [8], [9], [10]. Additionally, microorganisms present in bovine milk can serve as potential bioactive substances, contributing to the health of both offspring and adults [5], [11]. However, further research is required to gain a better understanding of the bovine milk microbiota. This entails conducting more comprehensive and systematic studies to explore the origins of the microbiota and their mechanisms of action in early-life gut development. This review offers insights into the sources of microorganisms in bovine milk and their potential roles in the initial gut colonization. This information will provide theoretical and experimental groundwork to enhance future applications of the bovine milk microbiota.

Table 1.

Nutritional composition of bovine milk.

Component (%)	Colostrum1	Transitional milk2			Mature milk3
		Milking 2	Milking 3	Milking 4
Total solids	23.9	17.9	14.1	13.9	12.9
Fat	6.7	5.4	3.9	4.4	4.0
Solids-not-fat	16.7	12.2	9.8	9.4	8.8
Protein	14.0	8.4	5.1	4.2	3.1
Lactose	2.7	3.9	4.4	4.6	5.0
Ash	1.11	0.95	0.87	0.82	0.74

Source: adapted from [140].

¹Colostrum: initial milk secretion postpartum.

²Transitional milk: milk from the second milking to the sixth milking (milking 2–4 are mentioned in the table).

³Mature milk: milk after the sixth milking, which is more stable in composition.

Microbiota composition in bovine milk

Previous studies on bovine milk microorganisms have primarily focused on their involvement in mastitis development and their impact. Therefore, it was previously believed that bovine milk obtained from healthy mammary glands was devoid of microorganisms. Milk samples containing microorganisms were attributed solely to environmental contamination sources [12] such as bedding, feces, barn air, milking utensils, and management practices [5], [7], [13], [14], [15], [16]. Efforts have been made to limit environmental contamination in milk samples. The studies of bovine milk microbiota involve three primary sampling methods: conventional aseptic sampling, milk-tank sampling, and teat cisternal puncturing. To briefly describe the conventional aseptic sampling method, the first streams of bovine milk are discarded for stimulation. Then, the teats are soaked in iodine tincture for 30 s and towel-dried. Next, another 2–3 milk streams are discarded, and the teats are wiped with 70% isopropyl alcohol. Once the teats are dry, another 2–3 streams of milk are discarded, and samples are collected [12], [17], [18], [19], [20], [21]. This method is extensively used in studies on bovine udder health. On the other hand, the milk-tank-sampling method is mostly employed to investigate the biosafety of raw bovine milk [8], [22], [23], [24], [25]. To further mitigate the impacts of the farm environment and sampling operations, a recent study employed an enhanced milk-sampling technique for directly collecting milk from mammary glands by inserting a vacuum syringe into the teat cistern [14]. Although no microbial growth was detected in the cultured cisternal samples, the analysis of 16S rRNA gene sequencing unveiled the presence of DNA that did not originate from the teat ducts or the external environment. Additionally, this study also yielded inconclusive results regarding the composition of microbiota in bovine milk due to variations in sampling methods. A recent review also mentioned that while genomes from several bacterial genera are routinely identified from samples of milk, and function of these organisms is uncertain as environmental factors have been shown to strongly influence the composition of these bacterial populations [26]. Consequently, further investigation is required to explore the potential sources and functions of these microorganisms.

Recent advancements in microbial analysis techniques, including 16S rRNA gene sequencing, metagenomics, transcriptomics, metabolomics, and culturomics have facilitated comprehensive and accurate detection of the milk microbiota. Numerous studies utilizing next-generation sequencing techniques have documented a wide range of microorganisms in bovine milk, specifically from healthy mammary glands, exhibiting site-specific variations [12], [14], [17], [18], [27], [28], [29]. Moreover, notwithstanding the absence of bacterial growth in certain milk samples during cultural analysis, several investigations have uncovered the presence of bacterial DNA originating from strictly anaerobic microorganisms, including Fusobacterium and Bacteroidetes. It is evident that these microorganisms could not have originated from the external environment [14], [30], [31], [32]. Standard aerobic culturing techniques demonstrate limitations in the identification of such microbial species.

Dominant microorganisms in bovine milk

The number of studies examining the microbiota in bovine milk has witnessed a substantial increase over the past decade. Researchers have gradually broadened their research scope, transitioning from primarily detecting pathogenic bacteria [12], [23], [31], [33] to exploring the entire microbial composition of bovine milk [7], [8], [10], [21], [22], [24], [27], [28], [29], [34], [35], [36], [37]. Within this microbial community, there exists a variety of bacteria, including both pathogenic strains capable of causing mastitis and milk spoilage, as well as probiotic strains such as Lactococcus, Streptococcus, Lactobacillus, Pediococcus, Leuconostoc, and Enterococcus species [6], [24]. In a study conducted by Hoque et al. [27], the analysis of milk samples from healthy cows using shotgun metagenomic sequencing revealed the presence of at least 146 bacterial strains. This unexpected complexity underscores the emerging significance of investigating the bovine milk microbiota as a distinct and crucial research field. The scientific community unanimously acknowledges that bovine milk harbors an exceptionally diverse and abundant microbial community.

Regardless of geographical area, season, lactation stage, and inter-individual variations among cows, specific microbial species in milk exhibit higher abundances [24], earning them the designation of dominant microorganisms, which constitute over 95% of the total relative abundance across all samples [22]. In a study conducted by Kim et al. [24] tested raw cow’s milk from 18 Korean farms using 16S rRNA gene sequencing was employed to analyze raw cow’s milk from 18 Korean farms, revealing four prevailing phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Similarly, both Ryu et al. [22] and Hoque et al. [27], employing metagenomic approaches, identified these same dominant phyla in bovine milk obtained from farms in Korean and Bangladeshi, respectively. Notably, Proteobacteria emerged as the most diverse and abundant taxonomic group in these studies. In contrast, a study conducted by Cremonesi et al. [10] focused on examining milk obtained from healthy Holstein Friesian and Rendena cows. Their findings revealed that the most prevalent phylum was Firmicutes, primarily represented by the genus Streptococcus, followed by Proteobacteria, Bacteroidetes, and Actinobacteria. Another investigation, encompassing the analysis of 33 phyla identified in bovine milk samples, indicated that the most abundant phyla were Firmicutes (40.8%), Proteobacteria (39.0%), Actinobacteria (9.40%), and Bacteroidetes (7.47%) [8]. Among the 785 genera detected in this study, the most abundant ones were Pseudomonas (19.6%), Bacillus (13.8%), Lactococcus (11.7%), and Acinetobacter (10.2%). In line with these findings, a separate study reported Firmicutes (57.7%) and Proteobacteria as the predominant microorganisms in healthy milk, collectively constituting over 90% of the microbial composition. The top four bacterial families identified in the samples were Ruminococcaceae, Enterobacteriaceae, Bacillaceae, and Pseudomonadaceae [21]. Significant microbial populations are also present in colostrum. Previous investigations focusing on colostrum composition have identified Proteobacteria (42%), Firmicutes (22%), and Bacteroidetes (21%) as the prevailing phyla [28], [38]. In the experimental trials performed by Lima et al. [29], it was demonstrated that Firmicutes (>40%) were the predominant phylum in bovine colostrum samples, followed by Proteobacteria, Bacteroidetes, Actinobacteria, Fusobacteria, and Tenericutes. Remarkably, this dominance was observed across colostrum samples regardless of parity. Derakhshani et al. [18], [19] also reported the prevalence of Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes as the most abundant phyla in bovine colostrum, constituting over 80% of the entire microbiota composition. Of particular note, the relative abundances of Firmicutes and Proteobacteria were almost equal (both approximately 44%-45%). Furthermore, Ruminococcaceae and Staphylococcus emerged as the most abundant family and genus, respectively, within this bacterial community.

Table 2 presents the relevant studies of dominant microorganisms in bovine milk. In summary, the dominant phyla were Proteobacteria and Firmicutes, accounting for approximately 90% of milk microorganisms. Proteobacteria included Acinetobacter, Pseudomonas, Escherichia, Vibrio, Erwinia, and Pantoea, and Firmicutes was dominated by Streptococcus, Enterococcus, Staphylococcus, and Bacillus. However, there is considerable variation observed in the bovine milk microbiota at different taxonomic levels, including order, family, genus, and species. Notably, Ruminococcaceae (Firmicutes) [19], [21], [29], [35], Bacillaceae [19], [21], [35], and Pseudomonadaceae (Proteobacteria, one of several taxa known to contaminate laboratory reagents) [10], [20], [21], [29] have been reported more frequently. Furthermore, researchers have identified a balanced relationship among microorganisms in bovine milk. For instance, there have been observed synergistic relationships between Pseudomonas/Propionibacterium and Lactobacillus/Bifidobacterium [8], [19]. In terms of relative abundance compared to total bacterial counts, Propionibacterium and Pseudoalteromonas tend to exhibit a negative correlation. These interactions between microbial groups may contribute to the maintenance of mammary health and milk quality [8].

Table 2.

Dominant microorganisms in milk from healthy cows.

Type of bovine milk1	Number of test cows	Sample collection	Methods for bovine milk microbiome analysis	Dominant bovine milk microbiota	Reference
Mature milk	Bull-tank milk	Conventional aseptic sampling2	16S rRNA gene sequencing (V4)	1) Phylum: Proteobacteria (32.94%-70.55%), Firmicutes (11.07%-39.19%), Bacteroidetes (10.56%-17.59%), and Actinobacteria (1.81%-6.87%).2) Genus: Pseudomonas, Lactococcus, Acinetobacter, Enterobacteriaceae, Bacillus, Bacteroides and Chryseobacterium.	[22]
Colostrum and transitional milk (0, 1, 2, and 6 d after calving)	9 Holstein cows	Conventional aseptic sampling	16S rRNA gene sequencing (V3-V4)	1) Phylum: Firmicutes (42.38%), Bacteroides (25.09%), Proteobacteria (22.86%), and Actinobacteria (7.64%).2) Genus: 35 genera, including Lactobacillus, Bacteroides, Escherichia_Shigella, Collinsella, Klebsiella, Alistipes, Clostridium, Prevotella, and Bifidobacterium.	[17]
Mature milk	7 Holstein cows	Conventional aseptic sampling	Whole metagenome sequencing	1) Phylum: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria (contributing to 96.51% of the total sequences).2) Genus: Acinetobacter (52.90%), Pseudomonas (22.81%), Micromonospora (10.57%), Eubacterium (5.37%), Catenibacterium (2.12%), and Ralstonia (0.12%).	[27]
Colostrum (immediately after calving)	15 Holstein cows	Taken from the bucket before first colostrum feeding (probably pasteurized)	16S rRNA gene sequencing (V3-V5)	1) The most dominant phylum: Proteobacteria (84.9%).2) The most dominant genus: Enhydrobacter (80.5%).	[38]
Mature milk	Bull-tank milk	Collected from the storage tanks of each farm	16S rRNA gene sequencing (V3-V4)	Genus: Pseudomonas (26.6%), Lactococcus (12.0%), Bacillus (11.0%), and Streptococcus (6.4%).	[23]
Mature milk	Not mentioned	Conventional aseptic sampling	16S rRNA gene sequencing (V3-V4)	Genus: Blautia (5.40%), Streptococcus (4.24%), Bradyrhizobium (3.30%), Sphingobacterium (3.05%), Clostridium (2.87%), Corynebacterium (2.69%), Alicyclobacillus (2.55%), Bacteroides (2.36%), Oscillospira (2.15%), and Treponema (2.14%).	[12]
Colostrum (immediately after calving) and mature milk	6 Holstein cows (HF) and 3 Rendena cows (REN)	Conventional aseptic sampling	16S rRNA gene sequencing (V3-V4)	1) Phylum: Firmicutes (HF 66%, REN 94%), Proteobacteria (HF < 13%, REN ∼ 1%), Bacteroidetes (HF < 8%, HF, REN ∼ 1%) and Actinobacteria (HF < 6%, REN ∼ 1%).2) Family: Streptococcaceae (HF 29.3%, REN 74.1%) and Lactobacillaceae (HF 6.9%, REN 14.0%); Ruminococcaceae, Bradyrhizobiaceae, Aerococcaceae and Staphylococcaceae in HF milk.3) Genus: Streptococcus (HF 27.5%, REN 68.6%); Bradyrhizobium, Staphylococcus and Corynebacterium in HF milk; Lactobacillus and Pediococcus in REN milk.	[10]
Colostrum and transitional milk (0, 1, and 6 d after calving)	54 Holstein cows	Conventional aseptic sampling	16S rRNA gene sequencing (V1-V2)	1) Phylum: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria.2) Order, family, and/or genus: Staphylococcus, Fusobacterium, Ruminococcaceae, and Clostridiales.	[18]
Colostrum (immediately after calving) and mature milk	11 Holstein cows	Conventional aseptic sampling	16S rRNA gene sequencing (V1-V2)	1) Phylum: Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria.2) Family and genus: Staphylococcus, Corynebacterium, Enterobacteriaceae, Pseudomonas, and Acinetobacter.	[19]
Mature milk	Bull-tank milk	Collected from the storage tanks of each farm	16S rRNA gene sequencing (V3-V4)	1) Phylum: Firmicutes (40.8%), Proteobacteria (39.0%), Actinobacteria (9.40%) and Bacteroidetes (7.47%).2) Genus: Pseudomonas (19.6%), Bacillus (13.8%), Lactococcus (11.7%) and Acinetobacter (10.2%).	[8]
Mature milk	14 Holstein cows	Conventional aseptic sampling	16S rRNA gene sequencing (V4)	1) Phylum: Firmicutes (57.7%) and Proteobacteria (26.0%).2) Family: Ruminococaceae, Enterobacteriaceae, Staphylococcaceae, Bacillaceae, Streptococcaceae, and Pseudomonadaceae.	[21]
Mature milk	20 Holstein cows	Conventional aseptic sampling and teat cisternal puncturing	16S rRNA gene sequencing (V4)	Not explicitly stated.	[14]
Transitional milk (4–6 d after calving) and mature milk	55 Holstein cows	Conventional aseptic sampling	16S rRNA gene sequencing (V4)	1) Phylum: Bacteroidetes.1) Family and genus: Acinetobacter, Cupriavidus, Janthinobacterium, Enhydrobacter, Staphlococcus, Corynebacterium, Fibrobacer, Aerococcaceae, Knoellia.	[20]
Mature milk	32 Holstein cows	Conventional aseptic sampling	16S rRNA gene sequencing (V1-V2)	1) Phylum: Proteobacteria (39.96%-48.30%), Firmicutes (30.25–40.28%), Bacteroidetes (8.38%-12.21%), and Actinobacteria (5.17%-11.29%).2) Genus: Streptococcus, Halomonas, Corynebacterium, Acinetobacter, and Atopostipes.	[141]
Colostrum (immediately after calving)	12 Holstein cows	Conventional aseptic sampling	16S rRNA gene sequencing (V3-V4)	1) Phylum: Proteobacteria (42%), Firmicutes (22%), and Bacteroidetes (21%).2) Genus: Lactococcus (13.6%), Pseudomonas (2.3%), Bacteroides (1.4%), Streptococcus (1.3%), and Staphylococcus (1.3%).	[28]
Mature milk	72 Holstein cows	Conventional aseptic sampling	16S rRNA gene sequencing (V4)	1) Phylum: Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Tenericutes, and Fusobacteria.2) Genus: Corynebacterium, Acinetobacter, Arthrobacter, Staphylococcus, and Psychrobacter.	[142]
Mature milk	12 Holstein cows	Conventional aseptic sampling	16S rRNA gene sequencing (V4)	Family: Ruminococcaceae (16.8%), Lachnospiraceae (7.0%), Aerococcaceae (6.8%), Enterobacteriaceae (6.3%), Planococcaceae (5.7%), Bacteroidaceae (5.4%), Corynebacteriaceae (5.1%), Clostridiaceae (4.2%), Bacillaceae (3.5%), and Staphylococcaceae (2.8%).	[35]
Mature milk	Bull-tank milk	Collected from the storage tanks of each farm	16S rRNA gene sequencing (V3-V4)	1) Phylum: Proteobacteria (25.85%-31.98%), Firmicutes (18.07%-24.13%), Actinobacteria (16.01%-17.39%), and Bacteroidetes (6.24%-6.99%).2) Genus: Massilia, Bacillus, Corynebacterium, Macrococcus, Staphylococcus, Arthrobacter, Streptococcus, and Burkholderia.	[24]
Mature milk	Bull-tank milk	Collected from the tanker trucks at 2 dairy processors	16S rRNA gene sequencing (V4)	1) Phylum: Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Tenericutes.2) Order, family and genus: Streptococcus (6.5%), Staphylococcus (5.4%), unclassified_Clostridiales (6.3%), Ruminococcaceae (4.3%), Corynebacterium (3.7%), Turicibacter (2.5%), Lachnospiraceae (2%), and Acinetobacter (1.2%).	[25]

¹Bovine colostrum refers to the initial milk secretion after delivery. Transitional milk usually represents milk from the second milking to 6 days postpartum, after which the milk is more stable in composition and is then referred to as mature milk ([140]).

²Conventional aseptic sampling follows the standard recommendations ([12], [14], [17], [18], [19], [20], [21]). Briefly, the first streams of bovine milk are discarded for stimulation. The teats are then soaked in iodine tincture for 30 s and towel-dried. Next, another 2–3 streams of milk are discarded, and the teats are wiped with 70% isopropyl alcohol. Once the teats are dry, another 2–3 streams of milk are discarded, and samples are collected.

Although the dominant microbial categories in bovine milk showed similarities across these studies (Table 2), their relative abundance ratios exhibited significant variations such as ratio of Proteobacteria to Firmicutes. These variations may have been influenced by factors, including geographic location, season, parity, lactation stage, physiological status, genetic effects, sampling, and analytical methods. A recent study reported the effects of subclinical mastitis and breed on the microbiota of milk, and showed that the milk bacterial microbiome was dominated by Firmicutes, Proteobacteria, and Actinobacteria, with an increase of Firmicutes in animals with subclinical mastitis and Proteobacteria in healthy animals and 45 and 51 discriminative taxonomic biomarkers associated with udder health status and with one of the four breeds respectively [39]. Moreover, studies have reported specific associations between milk-fat composition (e.g., saturated, monounsaturated, and polyunsaturated fatty acids) and dominant microbial groups (e.g., Bifidobacterium, Proteobacteria, Lactobacillus, Staphylococcus, and Streptococcus) [40], [41], [42], [43]. Additional investigation is warranted to elucidate the intricate interplay between the constituents of bovine milk and the microbial communities inhabiting the milk.

Core microorganisms in bovine milk

Similar to the dominant microbiota, the core microbiota exhibits a considerable degree of conservation, consistently appearing in at least 95% of samples, irrespective of individual, dietary, and environmental influences in cows [42], [44]. Nevertheless, in contrast to the dominant microbiota that comprises the uppermost 95% of microbial abundances, the core microbiota encompasses microorganisms with relatively lower abundances (≥1%) [22], [42], [44]. Nevertheless, due to some conceptual overlap, these two terms are frequently employed interchangeably in the current body of literature. In the context of plant research, Lemanceau et al. [45] have suggested that the core microbiota's definition should encompass the ecological niche functions as the fundamental unit of microbial functionality [46]. Consequently, there is a need for further refinement of the definition of core microbial groups present in maternal milk, taking into account their associated functions. Longitudinal studies should be conducted to delve deeper into identifying the essential functional microorganisms and to enhance the comprehension of the intricate associations between the core microbiota and its functional attributes. Metzger et al. [20] identified four culture-negative operational taxonomic units (OTUs), namely Faecalibacterium, Lachnospiraceae, Propionibacterium, and Aeribacillus, in all bovine milk samples obtained from healthy mammary glands. Furthermore, another study documented an eight-genera core microbial population in bovine milk, comprising Massilia, Bacillus, Corynebacterium, Macrococcus, Staphylococcus, Arthrobacter, Streptococcus, and Burkholderia [24]. Another previous study investigated the microbial diversity of over 400 quarter milk samples from 60 cows and found a high abundance of two bacterial families, Corynebacteriaceae and Staphylococcaceae, which were detected in all the cow udders and in more than 98% of quarter milk samples [47].

Research on the microbial profile of raw bovine milk before processing revealed a core milk microbiota comprising Streptococcus (6.5%), Staphylococcus (5.4%), and unidentified members of Clostridiales (6.3%) [25]. Thus, the core microbiota in bovine milk includes both beneficial genera (Lactobacillus, Lactococcus, and Propionibacterium) and genera commonly regarded as mastitic pathogens (Staphylococcus, Pseudomonas, and Streptococcus) [34]. While several studies have indicated inconsistencies in the compositions of the core bovine milk microbiota, other research has revealed that the functionality of these microbes remains relatively stable, despite the substantial variation in core microbial groups observed across geographic locations and individuals [48]. Given the potential physiological and metabolic significance of the microbial community in gut development and health [13], [17], [28], [49], it is imperative to identify the core microbiota to comprehend the stable and consistent components within intricate microbial assemblages. Conducting research on the core microbiota in bovine milk will facilitate a comprehensive understanding of the roles and functions of pivotal microorganisms in the microecosystem.

Although bacteria have been extensively studied as the core microbiota in maternal milk, it is important to note that maternal milk contains a wide range of microorganisms, including fungi, archaea, and viruses [3], [19], [27], [50], [51]. These microorganisms, alongside bacteria, form the core microbial network of breast milk, playing a crucial role in the health of both the mother and her offspring. Research has identified the presence of DNA and/or RNA from viruses, archaea, fungi, and protozoa in human milk [52], [53], [54]. However, when it comes to bovine milk, limited studies have explored microorganisms beyond bacteria, with our search yielding only two relevant studies [19], [27]. Derakhshani et al. [19] employed PCR amplification of eukaryotic ribosomal RNA genes to identify the presence of fungi; including Alternaria, Aspergillus, Candida, and Cryptococcus were present in bovine milk. On the other hand, Hoque et al. [27] utilized metagenomic analysis to investigate the compositions of microorganisms beyond bacteria, specifically focusing on viruses and archaea in bovine milk. Their findings revealed that Methanoplanus, Methanoculleus, Euryarchaeota, and Haloarcula were the most abundant archaeal genera in samples obtained from healthy mammary glands. The viral fraction of the current bovine milk microbiome is predominantly composed of the members from order Caudovirales, specifically the families Podoviridae, Siphoviridae, and Myoviridae families [27]. Techniques such as PCR amplification of eukaryotic ribosomal RNA genes and metagenomic analysis enable functional profiling of all microbial communities providing insights into microbial metabolism, virulence, and antibiotic resistance in bovine milk. Additionally, archaea, fungi, and viruses may have interrelationships with bacteria, potentially influencing the overall function of the bovine milk microbiota. Therefore, further studies are needed to determine the prevalence and role of fungi, viruses, and archaea in bovine milk.

Origins of the bovine milk microorganisms

The investigation into the origins of milk microorganisms has undergone significant expansion in recent decades. It is crucial to note that bovine milk microorganisms are not solely derived from the external environment. A recent study has highlighted substantial differences in microbial compositions between the mammary glands, udder skin, and the species shared between these two niches exhibited genotype variations [5], [55]. Furthermore, the presence of strictly anaerobic bacteria, such as Prevotella, Bacteroides, Faecalibacterium, Fusobacterium, and Clostridiales, in cow's milk suggests that the barn environment and other external factors cannot be considered as the primary sources of these bacteria [5], [12], [29]. Consequently, the origins of bovine milk microorganisms are more intricate than previously believed, potentially originating from both the surrounding environment and endogenous transfer (Fig. 1). In the case of endogenous transfer of maternal milk microorganisms, human studies have intensively investigated the retrograde pathway, which refers to the entry of infant oral microorganisms into the mammary glands [42], [56], [57], [58]. However, the retrograde pathway is restricted in dairy cows. In most of dairy farms, calves are separated from their dams shortly after birth, severely restricting the microbial interactions facilitated by maternal milk between dams and calves. As literature on the retrograde pathway of the bovine milk microbiota is limited, we will not delve into this pathway in this discussion. Instead, our focus will be on the endogenous pathways of the bovine milk microbiota, which encompass the entero-mammary and rumen-mammary pathways, as well as the resident microorganisms within the mammary glands. We will also address the controversies surrounding these pathways.

Fig. 1.

Potential origins of bovine milk microorganisms. External microbial sources of bovine milk microorganisms include drinking water, milking utensils, bedding, skin, feces, and barn environment [6], [7], [14], [15], [16]. Endogenous transfer pathways may consist of the entero-mammary [5], [13], [36] and rumen-mammary [20] pathways and mammary resident microorganisms [51], [72], [73]. Figure created with Microsoft PowerPoint and Procreate.

Entero-mammary and rumen-mammary pathways

In hosts, microbial communities in various niches do not act independently. They constantly interact and closely connect in their respective environments. Consequently, microorganisms present in the oral cavity, gastrointestinal tract, and other body sites are likely to enter the mammary glands and ultimately the maternal milk via endogenous pathways. Among these pathways, the entero-mammary pathway hypothesis has garnered significant attention (Fig. 2). Numerous studies conducted in humans and mice have provided evidence supporting the potential transfer of microbes from the intestines to the mammary glands [57], [59], [60], [61]. For instance, specific probiotics such as Limosilactobacillus fermentum CECT5716, Ligilactobacillus salivarius CECT5713, and Lactobacillus gasseri CECT5714 have been detected in breast milk following oral consumption [3], [7]. Similarly, oral administration of lactic acid bacterial strains to pregnant and lactating mice resulted in the detection of these strains in the mammary tissues and milk of the treated mice but not of the control mice [62], [63]. This finding provides additional evidence of the translocation of live microorganisms from the intestine to the mammary glands. Several studies [64], [65] have highlighted the abundance of monocytes, including dendritic cells and macrophages, in the intestines. Upon recognition of microbial surface antigens, these monocytes engulf the microorganisms and traverse the tight junctions without compromising the integrity of the intestinal epithelial barrier. Subsequently, the microorganism-laden monocytes can either be sequestered within the mesenteric lymph nodes or directly transported to the mammary glands via the blood-lymphatic system, eventually reaching maternal milk [64], [65]. A study has provided confirmation regarding the involvement of the blood-lymphatic system the transportation of microorganism. It was observed that administering Lactobacillus orally resulted in the detection of the same strain in the Peyer's patch cells of mice [63]. However, it remains uncertain whether this microbial translocation from the gastrointestinal tract to the mammary glands or maternal milk occurs selectively through immune cells. Further investigation is required to determine the underlying mechanisms involved. Previous study has indicated that intestinal dendritic cells and macrophages typically degrade phagocytosed bacteria before migrating to lymphatic tissues such as the spleen and lymph nodes. However, they also selectively retain and translocate limited numbers of live bacteria [7]. Hence, microorganisms that are resistant to degradation by monocytes may potentially be selectively transferred to the mammary glands. This suggests a potential mechanistic pathway through which microorganisms can be selectively transferred to the mammary glands [3], [5], [66].

Fig. 2.

Microbial transfer mechanism via the entero-mammary pathway. Commensal microbes in the gut are thought to be recognized and transported across the intestinal epithelium by monocytes such as dendritic cells and macrophages [3], [5], [36]. These microbe-carrying monocytes are either temporarily stored in the mesenteric lymph nodes or transferred directly to the mammary glands via the blood-lymphatic system [60], [63], [66]. Afterwards, via interactions with the mammary epithelium, these monocytes transport microorganisms into the mammary gland, then into the milk [3], [66]. Figure created with Microsoft PowerPoint and Procreate.

Limited research has been conducted on the entero-mammary pathway concerning the microbiota of bovine milk. The presence of anaerobic bacteria in both bovine colostrum and mature milk has sparked curiosity about their origin and the possibility of microbial translocation in dairy cows. A pioneering investigation by Young et al. [36] delved into the entero-mammary pathway hypothesis in lactating cows and provided evidence supporting the potential transfer of bacteria from the intestinal lumen to healthy mammary glands. The study observed the simultaneous presence of Ruminococcus, Bifidobacterium, and Peptostreptococcaceae in feces, milk and blood leukocytes of the same lactating cows. The aforementioned finding strongly suggests that intestinal microorganisms have the potential to be transported to bovine milk through circulating white blood cells [36]. However, further verification is required to confirm this hypothesis, and additional research is necessary to elucidate the specific mechanisms underlying the transportation of intestinal microorganisms to the mammary glands of cows.

Unlike monogastric animals such as humans and mice, ruminants possess a unique organ called the rumen, which serves as a site for microbial fermentation. The rumen houses a rich array of bacteria, protozoa, viruses, and fungi, contributing to its abundant and diverse microbiota. Interestingly, the rumen contents share certain similarities with bovine milk in terms of physical and chemical composition, including factors like pH and temperature [67], [68]. Moreover, both bovine milk and the contents of the rumen are rich in various nutrients that serve as substrates for microorganisms. This observation leads us to speculate that certain anaerobic microorganisms residing in the rumen could potentially be transferred to the mammary glands or bovine milk through a pathway known as the rumen-mammary pathway. This hypothesis gains support from previous study that have reported the presence of rumen microbiota DNA in bovine milk [20].

By considering these factors, we can deduce that there exists a plausible connection between the microbial populations in the rumen and those found in bovine milk. The shared nutrients and potential transfer of microorganisms from the rumen to the mammary glands highlight the possibility of an intricate interplay between the rumen and the mammary system in ruminants. Additional studies are necessary to investigate the feasibility of microbial translocation from the rumen to the mammary glands and subsequently into bovine milk.

Resident microorganisms in mammary glands

Maternal milk typically starts to accumulate in the mammary glands during late pregnancy. During this period, the entero-mammary pathway becomes more active, leading to a significant increase in the number of monocytes, including dendritic cells and macrophages, in the maternal intestines. Additionally, there is increased fetal compression against the mesenteric vessel, which facilitates the transit of intestinal microorganisms into the mammary glands [69]. Consequently, the number of microorganisms presents in the mammary glands and subsequently transferred to the maternal milk is significantly higher [51], [60], [69], [70]. Bacterial translocation from the gastrointestinal tract to the mammary glands is known to increase in pregnant and lactating rodents [40], [63]. Consequently, it is believed that the translocation of microorganisms to the mammary glands primarily occurs during late pregnancy and lactation, with limited translocation during non-pregnancy and non-lactation periods. However, some studies have discovered live microorganisms in the mammary tissues of women who have never breastfed, suggesting that the mammary glands may serve as a potential source of microorganisms in maternal milk [13], [71], [72], [73].

Metzger et al. [14] conducted a study to assess the impact of sampling technique on the bovine milk microbiota. Alongside conventional aseptic sampling, these authors collected bovine milk samples directly from the gland cistern by puncturing the udder. Surprisingly, PCR amplification of samples collected from gland cistern (83%) are higher than that of samples collected by conventional aseptic sampling, strongly indicating the presence of bacteria within the mammary glands [13]. This mammary microbiota could potentially originate from endogenous pathways including the entero-mammary pathway as microbial migration has been observed even in non-pregnant and non-lactating stages [51]. This process contributes to the development of original mammary gland microbiota. The original resident microbiota in the mammary glands could serve as another source of microorganisms that colonize milk, primary source of microorganisms when lactogenesis begins during late gestation [5], [10]. Similarly, if indigenous microorganisms exist within the mammary glands, they could stimulate plasma cells in the lamina propria to produce secretory immunoglobulin A (sIgA), which is typically present in low concentrations (0.1–0.2 mg/mL) in milk from cows with healthy mammary glands. This could be attributed to the relatively low abundance of microorganisms in bovine milk [30].

Controversy and prospects of the bovine milk microbiota arising from endogenous pathways

Although extensive research efforts have been made to elucidate the mechanism of intestinal microbial migration from the gut, the precise mode of interaction with the mammary epithelium upon their arrival, as well as the form in which the microorganisms reach the mammary glands (intracellular or extracellular, live or dead, intact or not) remains unclear. Organs that harbor commensal microbiotas, such as the intestines and skin are likely to exhibit limited responsiveness to elevated levels of microbe-associated molecular patterns (MAMPs) and display tolerance towards bacterial lipopolysaccharides (LPS) [74]. However, in the case of bovine mammary glands, the situation is different. Even low concentrations of LPS found in the intramammary bovine milk can lead to a significant influx of leukocytes. Moreover, MAMPs are injected into the bovine mammary glands through the teat duct induces a strong inflammatory response [75]. Furthermore, mammary epithelial cells possess toll-like receptors (TLRs) on their apical surface enabling them to detect microbial invasion and initiate inflammatory responses [30]. Therefore, these physiological responses do not seem to be indicative of the presence of microorganisms in the mammary glands. Furthermore, while the mammary epithelium is a component of the host secretory system, its classification as part of the mucosal system and its potential role in microorganismal transport through the mucosal system remain uncertain [3]. Moreover, the lactational physiology of humans and rodents differs significantly from that of ruminants. Some studies have indicated that unlike in humans and rodents, the migration of bovine lymphocytes between the intestines and mammary glands is limited [36], suggesting a weak entero-mammary link in ruminants. However, it remains unclear whether and to what extent this endogenous microbial circulation occurs in bovine physiology. Despite numerous studies explaining and validating entero-mammary translocation [5], [64], [65], [66], [76], the establishment of this pathway remains inconclusive. Further experimental evidence is needed to examine the mechanisms of the entero-mammary pathway. Further experimental evidence is needed to examine the mechanisms of the entero-mammary pathway, and clarify factors that can influence its composition, such as maternal body mass index and diet, use of antibiotics, time and type of delivery [77]. By clarifying and answering these questions, we can enhance our understanding of the origins of maternal milk microorganisms and confirm whether microorganisms are transported to the mammary glands via endogenous pathways or through a constant influx of exogenous microorganisms [3].

The development of multi-omics technologies has facilitated the significant in the field of culturomics and has brought about advancements in methods for avoiding, isolating, and validating microbial contamination at the strain level [51], [78], [79]. These developments present an opportunity to verify the existence of entero-mammary and rumen-mammary pathways, investigate the microbial communities residing in the mammary glands, and assess the respective contributions of endogenous and exogenous microbiotas to the composition of the bovine milk microbiota. Identifying the primary sources of the bovine milk microbiota is crucial for its manipulation and advancing bovine and human health. By utilizing these techniques, we can gain a deeper understanding of the complex interactions between different microbial populations within the bovine milk ecosystem.

Bovine milk microbiota affects early-life gut development

The gut is a vital organ that harbors a diverse microbial community and plays a crucial role in maintaining the overall health of host. Microorganisms have both direct and indirect impacts on nearly all physiological processes, including intestinal barrier function, energy metabolism, immune function, and even behavior [59], [80], [81]. During the delivery and early postnatal periods, neonates are exposed to a complex microbial community in the external environment, enabling the intergenerational transmission of the mammalian microbiota [80], [82]. Following birth, many infants are breastfed for a duration of up to 2 years, with exclusive breastfeeding typically lasting for approximately 6 months. This particular stage is critical for infant growth and establishment of the gut microbiota, exerting profound effects on short-term and lifelong physiological, metabolic, endocrinal, neurological, and immune functions [64], [81], [83]. It is a period characterized by rapid changes and highly dynamic processes [82], [84]. The intricate interactions between the gut microbiota and different host systems work play a vital role in influencing and supporting infant growth and development [82], [85]. Disruptions in the early microbial succession can have long-lasting and intergenerational consequences for infant growth and development, contributing to a range of conditions including obesity, allergies, asthma, diabetes, metabolic syndrome, and other chronic inflammatory diseases [64], [81], [86]. These outcomes underscore the significance of establishing a healthy and diverse gut microbiota during early life, as it serves as a foundation for optimal health and well-being throughout the lifespan.

Role of milk microorganisms in early gastrointestinal development

The early years of an infant's life are widely recognized as a critical period during which the gut microbiota undergoes significant changes, with early-life diet playing a prominent role in shaping the microbial composition and functionality [87], [88], [89]. Maternal milk holds paramount importance in the microbial programming of early life, as it provides essential nutrients for gut microbes and regulates their metabolic processes [85]. According to Calatayud et al. [87], breastfeeding emerges as the primary driver in the establishment of the gut microbiota in newborns, resulting in enduring differences in microbial profiles between breastfed and non-breastfed infants throughout their lives [86].

Breastfeeding plays a significant role in shaping the infant's metabolic, immune, and microbiological programming, thereby exerting a profound impact on their overall health, physical growth, and intellectual development [87], [88], [89]. During breastfeeding, there is a notable increase in the relative abundances and diversity of Bifidobacterium [84], [90], alongside an upregulation of genes associated with lipid and carbohydrate metabolism within the gastrointestinal tract of breastfed infants [83], [90]. These findings suggest a close association between maternal milk consumption and the composition and function of the infants' gastrointestinal microbiota. In addition to microbial transmission [13], [58], [85], [91], [92], [93], maternal milk also contributes to infants' gastrointestinal microbial composition and function through the transfer of non-microbial factors such as immunoglobulins, oligosaccharides, and growth factors [13], [85], [94]. However, various clinical factors such as prematurity, lack of milk, and mastitis can hinder timely provision of sufficient breast milk to some infants from their mothers. Consequently, this impedes the establishment of a healthy intestinal flora and compromises both the short-term and long-term health of the offspring [84]. Bovine milk, which is widely available on a commercial scale, serves as the primary alternative to human milk. While it cannot fully substitute for human milk, it can be used as a complementary food for infants during lactation to support their growth and development [4]. Interestingly, there are similarities in the microorganisms found in bovine milk and human breast milk. Studies have demonstrated an overlap in certain microorganisms found in both human and cow’s milk, including prominent taxa such as, Staphylococcus, Streptococcus, Pseudomonas, Bifidobacterium, Propionibacterium, Bacteroides, Corynebacterium, and Enterococcus [5], [7], [13], [58], [95], [96]. Additionally, it has been observed that Proteobacteria and Firmicutes are commonly found in both human and bovine milk [29], [58], [97]. The genera Staphylococcus and Streptococcus, which are universally prevalent in human milk [13], are also recognized as resident genera in bovine milk [24], [28]. Given these similarities, it is plausible that microorganisms present in bovine milk may fulfill similar functions to those of the human milk microbiota in infants. Furthermore, the beneficial microbiota found in bovine milk and dairy products have the potential to aid in the prevention or treatment of certain gastrointestinal disorders, thereby promoting a healthy and balanced microbial development in infants [50], [98].

The importance of the maternal milk microbiome in the establishment and development of the newborn gut microbiota has gained increasingly recognition (Fig. 3). Milk is now recognized as a reservoir of microorganisms [13], [50], [57], [76], [86], with a study estimating a viable bacterial density of 2–4 log colony-forming units/mL in human milk [99]. Consequently, maternal milk is thought to serve as a microbial selector, carrying a highly adaptive microbiome, known as the milk-oriented or milk-directed microbiota, which plays a crucial role in the colonization of infant guts. There is a notable association between the microbial compositions of newborn feces and mother's milk, with a shared genera accounting for 70%-88% of the total relative abundances in infant fecal samples [58]. Other studies examining the microbiota profiles of maternal milk and infant feces have discovered that 23%–33% of taxa, such as Streptococcus, Veillonella, Escherichia, Enterococcus, Lactobacillus, and Bifidobacterium, were present in both milk and fecal samples [49], [50]. Moreover, a metagenomic investigation revealed that 76% of the microbial species found in breastmilk were also present in the infant's gastrointestinal tract [91]. Thus, the milk microbiota may be vertically transmitted to the gastrointestinal tract of newborns, thereby playing a direct role in shaping the succession of microbial communities in infants' guts. However, the exact role of the maternal milk microbiota in establishing the microbial population in the infant gut and the specific mechanisms of its vertical transmission are still being explored. Continued research into the maternal milk microbiota and its impact on the establishment of the infant gut microbiome will provide a deeper understanding of early microbial colonization and potentially guide strategies aimed at promoting optimal gut health in newborns.

Fig. 3.

Potential mechanisms of the milk microbiota in infant intestinal development. Milk microorganisms may directly colonize the infant's gut [49], [50], [58], [85], [91], [92], [93] and play the following four roles. (1) They promote formation of an intestinal microecological niche by creating an anaerobic environment [13], [50], [93], [100], [101] and producing microbial metabolites [13], [64], [105] to facilitate specific microbial proliferation and inhibit colonization of pathogenic microbes. (2) They stimulate maturation of the innate and adaptive immune system and promote transition of the neonatal immune status from Th2 dominance to Th1/Th2 balance [105], [111], [112]. (3) They strengthen the intestinal epithelial barrier function by upregulating tight junctions and secreting associated cytoskeletal proteins [89], [106]. (4) Milk microorganisms may use cross-feeding to interact with milk components (e.g., oligosaccharides) and affect the gut microecological environment [49], [50], [85], [114], [115], [117], [121]. Figure created with Microsoft PowerPoint and Procreate.

The milk-oriented microbiota employs various mechanisms to influence establishment of a balanced gut environment. Maternal milk contains aerobic or facultative anaerobic microorganisms, including Enterobacteriaceae, Streptococcus, and Staphylococcus [7], [21], [24], [25], [29]. These microorganisms can capitalize on the remaining oxygen in the infant's gut, creating an anaerobic niche that fosters the proliferation of anaerobic bacteria such as Bifidobacterium, Bacteroides, and Clostridium [13], [50], [93], [100], [101]. Moreover, the majority of microorganisms present in milk engage in lactate metabolism, either through lactate synthesis or consumption [13]. This functional microbial community helps inhibit facultative and aerobic bacterial growth [71], [102], while preventing excessive lactate accumulation [103], [104]. Consequently, a homeostatic microecological environment is maintained in the intestine, promoting the construction of an active nutrient chain. Certain milk microorganisms, such as Bifidobacterium and Lactobacillus, utilize nutritional substrates in the gut to produce short-chain fatty acids (SCFAs), which exert beneficial effects on nutrition, interact with the intestinal barrier and impact immune function [64], [105].

In addition to their impact on the intestinal environment and the establishment of flora, milk microorganisms play crucial roles in maturation of immunity and the barrier function in gastrointestinal tracts of newborns. The maternal milk microbiota aids in the prevention of pathogen colonization and promotes the development of the mucosal defense system in neonatal intestines through mechanisms such as antimicrobial metabolite production and mucin production [89], [106]. Several commensal Staphylococcus species that colonized both maternal milk and the guts of breastfed infants possess adhesion-related genes [54], which may effectively compete with potential infections in infant guts [50]. Taxa such as Enterococcus and Lactobacillus are believed to contribute to the preparation of the newborn gut for optimal resistance against infections [107], [108], although the specific processes involved are not yet fully understood. A recent study demonstrated that mice supplemented with microencapsulated Lactiplantibacillus plantarum HM47 isolated from human breast milk significantly enhanced the intestinal lactic acid bacteria count and suppressed the enteric pathogenic bacterial count in mice [109], suggesting the Lactobacillus may compete microecological niche with pathogens in early-life gut. The exopolysaccharides secreted by the taxa may help to improve the adherence of lactic acid bacteria to the gut mucosa and inhibit the biofilm formation of pathogenic bacteria [110]. Additionally, milk microorganisms that colonize the intestinal mucosal layer can interact with the innate and adaptive immune systems, leading to enhanced intestinal competence and maturity in neonates [105], [111], [112]. Certain microorganisms, including Lactobacillus and Bifidobacterium, have the ability to activate natural killer cells, monocytes and macrophages, stimulate B-cell and T-cell differentiation, and subsequently induce production of specific antibodies and cytokines [105], [106]. The immunomodulating effects of Lactobacillus and Bifidobacterium from milk may also be attributed to their ability to produce exopolysaccharides, which have been shown to provide health benefits in antitumor, immunomodulatory activity, antioxidant activity and cholesterol-lowering properties [110]. Generally, these immune responses mentioned herein have the potential to facilitate the neonatal immune status, shifting it from Th2 dominance to a more balanced Th1/Th2 state. This transition also plays a vital role in the development of gut-associated lymphoid tissues (GALTs), encompassing crucial components such as Peyer’s patches and mesenteric lymph nodes [106], [113]. Consequently, these immune responses contribute significantly to diminishing the susceptibility to inflammatory diseases and immune-mediated pathological responses.

The microbiota present in maternal milk microbiota is believed to work synergistically with the inherent nutrients and bioactive constituents of the milk [114]. Metagenomic functional analysis conducted by van den Elsen et al. [85] has demonstrated the presence of prominent microorganisms in breast milk that possess metabolic functions related to carbohydrate, amino acid, and energy. It is hypothesized that milk microbial communities may be influenced by milk components, especially oligosaccharides, which have the potential to optimize the balance of intestinal microbiota through interactive processes and facilitate the colonization of specific microorganisms [50], [115]. However, the precise interplay between these milk components remains to be fully elucidated. Numerous beneficial milk microorganisms capable of utilizing oligosaccharides have been identified, including Bifidobacterium, Bacteroides, Lactobacillus, Collinsella, and Klebsiella [13], [49], [93], [116], [117], [118], [119], [120]. In a particular study the involvement of Streptococcus and Staphylococcus in the cross-utilization of oligosaccharides and metabolites was observed [121]; however, additional confirmation is required to validate these findings. These archetypical oligosaccharide degraders are naturally present in milk and play predominant role in the initial within the gastrointestinal tract of breastfed infants [117]. Since newborns lack the necessary enzymes to degrade milk oligosaccharides, they rely on the maternal milk microorganisms to catabolize the breakdown of these oligosaccharides into SCFAs [64]. This process facilitates the proliferation of beneficial microorganisms within the infant's gut which can be selectively induced.

Application of the bovine milk microbiota in early gut development

The microorganisms present in bovine milk play a crucial role in modulating modulate and maintaining dynamic homeostasis within the gut microbiotas of infant, promoting their development in a beneficial manner. Additionally, bovine milk has the ability to influence the establishment of microbial populations in the gastrointestinal tract and oral cavity of calves [122]. In a study exploring the correlation between the maternal microbiota and early successional development of the gastrointestinal tract microbiome in calves, it was discovered that approximately 10.6% and 9.6% OTUs in colostrum were shared with the luminal and mucosal microbiotas of the calves, respectively. This finding suggests that colostrum contributes to the composition of the gastrointestinal tracts in calves [13], [28]. Hence, bovine milk has the potential to serve as a vehicle for delivering probiotics to the human gut through dietary means. In support of this, Aljutaily et al. [123] observed that a diet supplemented with probiotic-enriched cow cheese led to alterations in the gut microbiotas of mice characterized by increased relative abundances of the order Clostridiales, family Ruminococcaceae, and Lachnospiraceae. Furthermore, research findings have indicated that fermented dairy products possess the ability to modulate the fecal microbiota and reduce markers of systematic inflammation [124], [125], [126], [127]. Research on young rat found that supplementation with milk based on standard diet could increase relative abundance of Bifidobacteriaceae, compared with whilst soy and almond beverage [128]. Numerous extensive studies have demonstrated that the consumption of bovine milk can enhance the intestinal microbial activity in humans, mice, and rats by promoting growth of bacteria that produce SCFAs such as Blautia, Roseburia, Bacteroides, Streptococcus, and Alloprevotella [123], [129], [130], [131].

Probiotics derived from bovine milk have been extensively studied in human and employed to influence the relative abundances of gut bacteria. These probiotics primarily consist of Lactobacillus and Bifidobacterium, both of which have a well-established history of safe usage [76]. Notably, research has demonstrated that specific Lactobacillus species, including Limosilactobacillus reuteri, Limosilactobacillus fermentum, Lacticaseibacillus casei, and Ligilactobacillus salivarius, can effectively modulate composition and diversity gut microbiota, thereby enhancing the immune system [127], [132]. Moreover, evidence supports the utilization of Bifidobacterium from bovine milk as probiotics. A study has documented that Bifidobacterium can be beneficial in the treatment of conditions such as infant diarrhea, rotaviral infection, and murine colorectal cancer [50]. Additionally, Enterococcus faecium isolated from bovine milk has been identified as a potentially safe probiotic [133]. The actions of this product include inhibiting the growth of pathogenic microorganisms such as Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Shigella dysenteriae, and Streptococcus agalactiae. These findings suggest that bovine milk products enriched with probiotics can be highly effective in promoting the development of a healthy gut microbiome. However, the precise mechanism through which these probiotics derived from bovine milk impact gut development of infants remains unclear, and warranted further research. Moreover, bovine milk can be regarded as a natural symbiotic food that inherently encompasses both probiotics and prebiotics within its original components, thereby fostering a symbiotic relationship among microbes in the human diet [116], [118], [123]. Understanding these characteristics of bovine milk is crucial for discerning the ways in which this natural, nutritious food supports the development and maturation of infant gastrointestinal systems. However, the precise mechanism by which microorganisms in milk contribute to this intricate process remains unclear.

Limitations and prospects of using the bovine milk microbiota

Despite the multiple advantages of bovine milk microorganisms in the infant gut microenvironment, there are still some obstacles to their application. One of the main obstacles in milk processing is the heat treatment of raw milk. Raw milk, which undergoes no processing after milking, serves as an ideal nutritional medium for microbial growth. Consequently, during the stages of collection, storage, and transportation, numerous harmful microorganisms can multiply and produce toxic metabolites that pose a threat to human health. Therefore, milk is often subjected to thermal processes after collection from cows to kill pathogenic microorganisms and improve its biosafety before being supplied for human consumption. However, these thermal processes can also disrupt the original microbiota in bovine milk, thus greatly reducing microbial abundance and diversity. Pasteurization and ultra-high temperature (UHT) treatment are the two most common approaches for heat processing of bovine milk. Both pasteurization and UHT could kill pathogens, though the two methods differ in processing time and temperature. Pasteurization treats milk at either 72–75 ℃ for 15 s (higher heat, shorter time) or 62–65 ℃ for 5 min (lower heat, longer time). Conversely, UHT treats milk at a very high temperature (130–140 ℃) for 2–3 s [134], [135]. While both methods destroy most pathogenic microbes, ensure the microbiological safety of milk, and prevent dairy-borne diseases in humans, they can negatively affect flavor, bioactive compounds, and beneficial microorganisms. Compared with UHT, pasteurization is gentler and has less impact on milk's bioactive components and microorganisms; however, apart from killing or inhibiting the most harmful microbes, pasteurization eliminates most beneficial microorganisms and damages the bioactive components. Compared with raw bovine milk, pasteurization reduces the diversity and vastly alters the milk microbiota composition. For example, although Proteobacteria and Firmicutes remained the dominant phyla, the abundances of Firmicutes, Bacteroidetes, and Actinobacteria decreased, while Pseudomonas, Streptococcus, and Cyanobacteria increased after pasteurization [114].

To avoid thermal damage to naturally beneficial microorganisms, more research is warranted on how to better harness these microorganisms in bovine milk to support human health. -For instance, Beneficial microorganisms in bovine milk can be retained or reconstructed by optimizing the milking process, improving thermal processing, and re-culturing the milk microbiota to maximize its biological value. Strategies to recover the maternal milk microbiota have been investigated in humans [136], [137], [138]. Researchers have discovered that incubating pasteurized breast milk with 10%-30% of unpasteurized breast milk can partially restore the natural microbiological and metabolomic profiles in pasteurized breast milk [137]. However, no similar research has been published on the effects of mixing unpasteurized and pasteurized bovine milk on microbial composition. Additionally, exploration of the beneficial microorganisms in bovine milk and their potential as a natural probiotic reservoir is needed. On the basis of the reported benefits of the bovine milk microbiota in promoting gut development and homeostasis, isolation of potential probiotic strains from bovine milk remains a top priority for investigators. Nevertheless, the identification and isolation of potential probiotic strains have predominantly focused on a select few traditional bacterial species, primarily Bifidobacterium and Lactobacillus, due to their established safety and efficacy in the nutritional and health sectors [76], [139]. Exploring unconventional bacterial species through research may unveil additional viable probiotics that can enhance gut health. Furthermore, advancements in methodologies and research techniques offer an opportunity to gain a comprehensive understanding of the functions and contributions of the commensal microbiota present in bovine milk, thereby promoting optimal gut development and immunological functionality.

Conclusions

Milk derived from dairy cows serves as a prominent part for human diet, particularly infants and children. Within bovine milk, there exists a diverse array of microorganisms that play crucial roles in fostering the development of the gastrointestinal tract and facilitating immune function maturation in offspring. Interestingly, these milk-derived microorganisms can yield comparable effects to those found in human breast milk. Previous research efforts have predominantly focused on investigating the impact of pathogenic and conditionally pathogenic microbes on udder health and the quality of dairy products. However, limited attention has been given to exploring the influence of bovine milk microorganisms on the development of the infant gut. Thus, it is imperative to elucidate the composition and origins of natural microorganisms present in bovine milk to pave the way for their potential applications. Existing research comparing the microbial compositions of bovine milk and human breast milk has identified similarities, suggesting that the bovine milk microbiota may play a role in modulating the establishment and maturation of infant gut microecosystems in a similar manner to the human milk microbiota. Nevertheless, challenges arise in utilizing the bovine milk microbiota, including potential disruptions caused by pasteurization or UHT treatments. To fully explore the benefits offered by the bovine milk microbiota, it is crucial to employ enhanced sampling techniques and advanced multi-omics analytical approaches. To comprehensively understand the microbial functions and vertical transfer mechanisms of the bovine milk microbiota, well-designed experimental studies are warranted. These studies will contribute significantly to advancing our knowledge and appreciation of the bovine milk microbiota and its potential applications in various domains.

Funding

The present research was supported by grants from the National Key research and Development Program of China (2021YFF1000703-03) and the National Natural Science Foundation of China (32272902).

Compliance with ethics requirements

This review doesn’t involve the use of human and animal subjects, thus no ethics statement is provided.

CRediT authorship contribution statement

Wenli Guo: Conceptualization, Writing – original draft, Writing – review & editing. Shuai Liu: Conceptualization, Writing – original draft, Writing – review & editing. Muhammad Z. Khan: Writing – review & editing, Visualization. Jingjun Wang: . Tiyuan Chen: . Gibson M. Alugongo: Writing – review & editing, Visualization. Shengli Li: Writing – review & editing, Visualization. Zhijun Cao: Supervision, Conceptualization, Funding acquisition, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Wenli Guo received her bachelor’s degree in 2020 from China agriculture university, followed by Ph.D. work at the same college in Dr. Cao’s lab. Her research is closely related to bovine milk bioactive compounds and their mechanism in early-life gut development, hoping to find new effective ways to prevent or cure gastrointestinal diseases of dairy calves.

Dr. Shuai Liu serves as a postdoctor in Dr. Cao’s lab, China agricultural university, since he received his Ph.D. in 2022. His research field is focused on intestinal microbiome development of early life in dairy cattle. Up to now, he has published 5 peer-reviewed articles in JCR Q1 journals, and revealed how nutrients affect the microbiome and immune development of dairy calves.

Dr. Muhammad Zahoor Khan is currently working as an assistant Professor in Department of Animal Sciences, university of Agriculture Dera Ismail Khan, Pakistan. In 2019, he got his PhD degree in Animal Breeding and Genetics (Nutrigenomics) from China Agricultural University Beijing China. He worked for almost three years as a collaborative researcher with State Key Laboratory of Animal Nutrition, Beijing Engineering Technology Research Center of Raw Milk Quality and Safety Control, College of Animal Science and Technology, China Agricultural University, Beijing, China. Dr. Zahoor has already published more than 40 articles in well-reputed Journals i.e Animal Nutrition, Frontiers in Immunology, Journal of Dairy science and Journal of Animal Science and Technology etc.

Jingjun Wang is a PhD candidate in Dr. Cao’s lab at China Agricultural University. His research fields are data mining for dairy production systems and environment sustainability. He also works as the associate editor of Hoard’s Dairyman China.

Tianyu Chen is a PhD candidate in Dr. Cao's Lab, China agricultural university. His research field is focused on calf nutrition and feed efficiency. Up to now, as first author and co-first author, he has published 3 papers, and revealed effects of hay supplementation on calves.

Dr. Gibson Maswayi Alugongo received his M.D. and Ph.D. in Dr. Cao’s lab, China agricultural university, respectively in 2015 and 2020. Subsequently, he worked as a post-doctor from 2020 to 2022, and focused on dairy cattle nutrition. He awarded the Chinese Government Outstanding International Student Scholarship. Until now, He conducted more than 5 in vivo experiment in farms and has published 4 peer-reviewed articles.

Dr. Shengli Li obtained his doctorate degree in animal nutrition science from China Agricultural University in July 1996. Since 2003, Dr. Li has been with China Agricultural University, working as an assistant professor and professor in dairy cattle nutrition. Dr. Li is currently the vice-director (Animal Nutrition) of the State Key Laboratories, chief scientist in national dairy products industry technology system, a specialist to the China School Milk Programme. Dr. Li was awarded the first prize for Chinese Agricultural Science awarded by the Ministry of Agriculture in 2013 and the second prize of National Scientific and Technological Progress Award in 2014. He has published more than 60 articles in peer-reviewed journals.

Dr. Zhijun Cao received his Ph.D. from CAU in 2007, and since then he worked as an assistant professor, associate professor and professor in college of animal science and technology, China agricultural university. Dr Cao's laboratory focuses on Dairy cattle nutrition and milk quality, particularly the GIT development of dairy cows and calves. He has published more 50 peer-reviewed articles in Microbiome, Journal of Dairy Science and Animal Nutrition. In 2011, Dr. Cao launched Elite Cattleman Program in order to cultivate future stars for dairy industry, and more then 3000 students benefited from this program so far. In 2016, Dr. Cao founded International Calf and Heifer Organization, which aims to integrate and utilize global resources, and eventually contribute to the sustainable development of dairy industry. Dr. Cao has received a number of honors and awards, including the Distinguished Professor of Talent Program of the Ministry of Education (MOE), the first prize of Scientific and Technological Progress Award (MOE), etc.