The detailed analysis of the microbiome and resistome of artisanal blue-veined cheeses provides evidence on sources and patterns of succession linked with quality and safety traits.

BACKGROUND: Artisanal cheeses usually contain a highly diverse microbial community which can significantly impact their quality and safety. Here, we describe a detailed longitudinal study assessing the impact of ripening in three natural caves on the microbiome and resistome succession across three different producers of Cabrales blue-veined cheese. RESULTS: Both the producer and cave in which cheeses were ripened significantly influenced the cheese microbiome. Lactococcus and the former Lactobacillus genus, among other taxa, showed high abundance in cheeses at initial stages of ripening, either coming from the raw material, starter culture used, and/or the environment of processing plants. Along cheese ripening in caves, these taxa were displaced by other bacteria, such as Tetragenococcus, Corynebacterium, Brevibacterium, Yaniella, and Staphylococcus, predominantly originating from cave environments (mainly food contact surfaces), as demonstrated by source-tracking analysis, strain analysis at read level, and the characterization of 613 metagenome-assembled genomes. The high abundance of Tetragenococcus koreensis and Tetragenococcus halophilus detected in cheese has not been found previously in cheese metagenomes. Furthermore, Tetragenococcus showed a high level of horizontal gene transfer with other members of the cheese microbiome, mainly with Lactococcus and Staphylococcus, involving genes related to carbohydrate metabolism functions. The resistome analysis revealed that raw milk and the associated processing environments are a rich reservoir of antimicrobial resistance determinants, mainly associated with resistance to aminoglycosides, tetracyclines, and ß-lactam antibiotics and harbored by aerobic gram-negative bacteria of high relevance from a safety point of view, such as Escherichia coli, Salmonella enterica, Acinetobacter, and Klebsiella pneumoniae, and that the displacement of most raw milk-associated taxa by cave-associated taxa during ripening gave rise to a significant decrease in the load of ARGs and, therefore, to a safer end product. CONCLUSION: Overall, the cave environments represented an important source of non-starter microorganisms which may play a relevant role in the quality and safety of the end products. Among them, we have identified novel taxa and taxa not previously regarded as being dominant components of the cheese microbiome (Tetragenococcus spp.), providing very valuable information for the authentication of this protected designation of origin artisanal cheese. Video Abstract.

Pasture feeding improves the nutritional, textural and techno-functional characteristics of butter.

There is an increasing consumer desire for pasture-derived dairy products, as outdoor pasture-based feeding systems are perceived as a natural environment for animals. Despite this, the number of grazing animals globally has declined as a result of the higher milk yields achieved by indoor, total mixed ration feeding systems, in addition to the changing climatic conditions and lower grazing knowledge and infrastructure. This has led to the development of pasture-fed standards, stipulating the necessity of pasture and its minimum requirements as the primary feed source for products advertising such claims, with various requirements depending on region for which it was produced. This work investigates the differences in the composition and techno-functional properties of butters produced from high, medium and no pasture allowance diets during early, mid and late lactation. Butters were produced using milks collected from 3 feeding systems: outdoor pasture grazing (GRS; high pasture allowance); indoor total mixed ration (TMR; no pasture allowance); and a partial mixed ration (PMR; medium pasture allowance) system, which involved outdoor pasture grazing during the day and indoor TMR feeding at night. Butters were manufactured during early, mid and late lactation. Creams derived from TMR feeding systems exhibited the highest milk fat globule size. The fatty acid profiles of butters also differed significantly as a function of diet, and could be readily discriminated by partial least squares analysis. The most important fatty acids in such analysis, as indicated by their highest variable importance projection scores, were CLA C18:2 cis-9 trans-11 (rumenic acid), C16:1 n-7 trans (trans-palmitoleic acid), C18:1 trans (elaidic acid), C18:3 n-3 (α-linolenic acid) and C18:2 n-6 (linoleic acid). Increasing pasture allowances resulted in reduced crystallization temperatures and hardness of butters, while concurrently increasing the 'yellow' b* color. Yellow color was strongly correlated with Raman peaks commonly associated with carotenoids. The milk fat globule size of cream decreased with advancing stage of lactation and churning time of cream was lowest in early lactation. Differences in the fatty acid and triglyceride contents of butter as a result of lactation and dietary effects demonstrated significant correlations with the hardness, rheological, melting and crystallization profiles of the butters. This work highlighted the improved nutritional profile and functional properties of butter with increasing dietary pasture allowance, primarily as a result of increasing proportions of unsaturated fatty acids. Biomarkers of pasture feeding (response in milk proportionate to the pasture allowance) associated with the pasture-fed status of butters were also identified as a result of the significant changes in the fatty acid profile with increasing pasture allowance. This was achieved through the use of 3 authentic feeding systems with varying pasture allowances, commonly operated by farmers around the world and conducted across 3 stages of lactation.

Impact of feeding regimes and lactation stage on sensory attributes of Cheddar cheese.

This study investigated the effects of diet and stage of lactation (SOL) on sensory profiles, texture, volatile profiles, and colour of Cheddar cheese. Cheddar cheese was manufactured from early-, mid-, and late-lactation milk obtained from seasonally calved cows (n = 54). Cows were assigned a diet; group 1: perennial ryegrass (GRS), group 2: total mixed ration (TMR), and group 3: partial mixed ration (PMR). Instrumental analysis was performed at 270 days (mature Cheddar). Sensory evaluation took place after 548 days (extra mature Cheddar). Toluene was the only volatile compound that was significantly influenced by diet. The trained panel rated early-lactation cheese as stronger than mid- and late- for cowy/barny flavour and late-lactation cheese as sweeter than early- and mid-lactation cheese. Mid-lactation cheese was liked least overall. Early-lactation cheeses were rated higher for 'crumbly' texture than mid- and late. Diet affected consumer ratings, with GRS and PMR cheese rated as more intense than TMR for flavour, aftertaste, and saltiness. Consumers reported that TMR cheese was lighter in colour compared to GRS cheese, which was supported by instrumental analysis. Consumers perceived GRS as more springy and less crumbly than TMR and PMR, while Texture Profile Analysis indicated that TMR was harder than GRS. Consumer segmentation was observed with two clear preference groups, one preferring GRS and one preferring TMR. For both groups, 'taste' seemed to be the main driver of liking, highlighting that consumer preference is most impacted by individual taste preferences.

Impact of varying levels of pasture allowance on the nutritional quality and functionality of milk throughout lactation.

The objective of this study was to examine the impact of increasing proportions of grazed pasture in the diet on the composition, quality, and functionality of bovine milk across a full lactation. Fifty-four spring-calving cows were randomly assigned to 1 of 3 groups (n = 18), blocked on the basis of mean calving date (February 15, 2020 ± 0.8 d), pre-experimental daily milk yield (24.70 ± 3.70 kg), milk solids yield (2.30 ± 0.27 kg), lactation number (3.10 ± 0.13), and economic breeding index (182 ± 19). Raw milk samples were obtained weekly from each group between March and November 2020. Group 1 (GRS) consumed perennial ryegrass and was supplemented with 5% concentrates (dry matter basis); group 2 was maintained indoors and consumed a total mixed ration (TMR) diet consisting of maize silage, grass silage, and concentrates; and group 3 consumed a partial mixed ration diet (PMR), rotating between perennial ryegrass during the day and indoor TMR feeding at night. Raw milk samples consisted of a pooled morning and evening milking and were analyzed for gross composition, free amino acids, fatty acid composition, heat coagulation time, color, fat globule size, and pH. The TMR milks had a significantly higher total solids, lactose, protein, and whey protein as a proportion of protein content compared with both GRS and PMR milks. The GRS milks demonstrated a significantly lower somatic cell count (SCC), but a significantly higher pH and b*-value than both TMR and PMR milks. The PMR milks exhibited significantly lower total solids and fat content, but also demonstrated significantly higher SCC and total free amino acid content compared with GRS and TMR. Partial least squares discriminant analysis of fatty acid profiles displayed a distinct separation between GRS and TMR samples, while PMR displayed an overlap between both GRS and TMR groupings. Variable importance in projection analysis identified conjugated linoleic acid cis-9,trans-11, C18:2n-6 cis, C18:3n-3, C11:0, and C18:2n-6 trans as the largest contributors to the variation between the diets. Milk fats derived from GRS diets exhibited the highest proportion of unsaturated fats and higher unsaturation, health-promoting, and desaturase indices. The lowest proportions of saturated fats and the lowest atherogenic index were also exhibited by GRS-derived milk fats. This work highlights the positive influence of grass-fed milk for human consumption through its more nutritionally beneficial fatty acid profile, despite the highest milk solid percentages derived from TMR feeding systems. Furthermore, this study demonstrates the proportional response of previously highlighted biomarkers of pasture feeding to the proportion of pasture in the cow's diet.

The Protein Composition and In Vitro Digestive Characteristics of Animal- versus Plant-Based Infant Nutritional Products.

The protein composition and digestive characteristics of four commercially available infant formulae (IF) manufactured using bovine (B-IF), caprine (C-IF), soy (S-IF), and rice (R-IF) as a protein source were examined in this study. Plant-based formulae had significantly higher crude protein and non-protein nitrogen (NPN) concentrations. Static in vitro gastrointestinal digestion of these formulae, and subsequent analysis of their digestates, revealed significantly higher proteolysis of B-IF at the end of gastrointestinal digestion compared to the other formulae, as indicated by the significantly higher concentration of free amine groups. Furthermore, differences in structure formation during the gastric phase of digestion were observed, with formation of a more continuous, firmer coagulum by C-IF, while R-IF demonstrated no curd formation likely due to the extensive hydrolysis of these proteins during manufacture. Differences in digestive characteristics between formulae manufactured from these different protein sources may influence the bio-accessibility and bioavailability of nutrients, warranting additional study.

Compositional and functional properties of milk and dairy products derived from cows fed pasture or concentrate-based diets.

Worldwide milk production is predominantly founded on indoor, high-concentrate feeding systems, whereas pasture-based feeding systems are most common in New Zealand and Ireland but have received greater attention recently in countries utilizing conventional systems. Consumer interest in 'pasture-fed' dairy products has also increased, arising from environmental, ethical, and nutritional concerns. A substantial body of research exists describing the effect of different feeding strategies on the composition of milk, with several recent studies focusing on the comparison of pasture- and concentrate-based feeding regimes. Significant variation is typically observed in the gross composition of milk produced from different supplemental feeds, but various changes in the discrete composition of macromolecular components in milk have also been associated with dietary influence, particularly in relation to the fatty acid profile. Changes in milk composition have also been shown to have implications for milk and dairy product processability, functionality and sensory properties. Methods to determine the traceability of dairy products or verify marketing claims such as 'pasture-fed' have also been established, based on compositional variation due to diet. This review explores the effects of feed types on milk composition and quality, along with the ultimate effect of diet-induced changes on milk and dairy product functionality, with particular emphasis placed on pasture- and concentrate-based feeding systems.

Invited review: A 2020 perspective on pasture-based dairy systems and products.

Grazing pasture is the basis for dairy production systems in regions with temperate climates, such as in Ireland, New Zealand, parts of Australia, the United States, and Europe. Milk and dairy products from cows on pasture-based farms predominantly consuming fresh grazed grass (typically classified as "grass-fed" milk) have been previously shown to possess a different nutrient profile, with potential nutritional benefits, compared with conventional milk derived from total mixed ration. Moreover, pasture-based production systems are considered more environmentally and animal welfare friendly by consumers. As such, there is significant potential for market capitalization on grass-fed dairy products. As competition in this space increases, the regulations of what constitutes as grass-fed vary between different regions of the world. With this in mind, there is a need for clear and independently accredited grass-fed standards, defining the grass-fed criteria for labeling of products as such, subsequently increasing the clarity and confidence for the consumer. This review outlines the numerous effects of pasture production systems on dairy product composition, nutritional profile, and sustainability, and highlights potential future methods for authentication.

The Impact of Seasonality in Pasture-Based Production Systems on Milk Composition and Functionality.

Seasonal calving, pasture-based dairy systems are widely practiced in countries with a temperate climate and plentiful rainfall such as Ireland and New Zealand. This approach maximizes milk production from pasture and, consequently, is a low-cost, low-input dairy production system. On the other hand, the majority of global milk supply is derived from high input indoor total mixed ration systems where seasonal calving is not practiced due to the dependence on ensiled silages, grains and concentrated feeds, which are available year-round. Synchronous changes in the macro and micronutrients in milk are much more noticeable as lactation progresses through early, mid and late stages in seasonal systems compared to non-seasonal systems-which can have implications on the processability and functionality of milk.

Milk fat: opportunities, challenges and innovation.

Milk fat is a high-value milk component that is processed mainly as butter, cheese, cream and whole milk powder. It is projected that approximately 35 million tonnes of milk fat will be produced globally by 2025. This surplus, enhances the need for diversification of milk fat products and the milk pool in general. Infant milk formula producers, for instance, have incorporated enzyme modified ("humanised") milk fat and fat globule phospholipids to better mimic human milk fat structures. Minor components like mono- and di-glycerides from milk fat are increasingly utilized as emulsifiers, replacing palm esters in premium-priced food products. This review examines the chemistry of milk fat and the technologies employed for its modification, fractionation and enrichment. Emerging processing technologies such as ultrasound, high pressure processing, supercritical fluid extraction and fractionation, can be employed to improve the nutritional and functional attributes of milk fat. The potential of recent developments in biological intervention, through dietary manipulation of milk fatty acid profiles in cattle also offers significant promise. Finally, this review provides evidence to help redress the imbalance in reported associations between milk fat consumption and human health, and elucidates the health benefits associated with consumption of milk fat and dairy products.

A Cross-Cultural Evaluation of Liking and Perception of Salted Butter Produced from Different Feed Systems.

Perception and liking among Irish, German and USA consumers of salted butter produced from different feed systems-outdoor grass (FS-GRSS), grass/clover (FS-CLVR), and indoor concentrate (FS-TMR)-was investigated. A consumer study was conducted in all three countries. Irish and German assessors participated in ranking descriptive analysis (RDA), whereas descriptive analysis (DA) was carried out by a trained panel in the USA. Volatile analysis was conducted to identify differences in aroma compounds related to cow diet. Overall, there was no significant difference in overall liking of the butters, among USA, German and Irish consumers, although cross-cultural preferences were evident. Sensory attribute differences based on cow diet were evident across the three countries, as identified by German and Irish assessors and trained USA panelists, which are likely influenced by familiarity. The abundance of specific volatile aromatic compounds, especially some aldehydes and ketones, were significantly impacted by the feed system and may also contribute to some of the perceived sensory attribute differences in these butters.

Effect of Pseudomonas fluorescens proteases on the quality of Cheddar cheese.

The objective of this study was to investigate the effect of adding different levels of a thermoresistant protease produced by a Pseudomonas fluorescens strain to milk on the manufacture and quality of Cheddar cheese. Fresh raw milk was collected, standardized, and pasteurized at 72°C for 15 s, and the enzyme was added to give a protease activity of 0.15 or 0.60 U/L (treatments P1 and P4, respectively), while one sample had no enzyme added (control). Milk was stored at 4°C for 48 h and Cheddar cheese was manufactured after 0 and 48 h of storage. Results indicated that the protease was active in milk during 48 h of storage; however, its effect on milk composition was minimal. The protein that was preferentially hydrolyzed by the protease over storage was ß-casein, followed by κ-casein. The mean cheese yield and recovery of fat and protein obtained for all cheeses were not affected by protease activity. The protease showed low activity during cheese manufacture, possibly because of unfavorable conditions, including low pH. One of the factors that might have influenced protease activity was the pH of the curd (approximately 6.55 after acidification and 5.35 at milling), which was lower than that at which the enzyme would have optimum activity (pH 7 to 9). Consequently, the composition, pH, patterns of proteolysis, and hardness of all cheeses produced were similar and in accordance with values expected for that type of cheese, independently of the protease activity level. However, slight increases in proteolysis were observed in P4 cheeses and produced using milk stored for 48 h. Both the P1 and P4 cheeses had higher concentrations of free amino acids (FAA) compared with the control, whereas urea-PAGE electrophoretograms indicated a greater breakdown of caseins in the P4 cheese samples, which may be related to possible increases in numbers of proteolytic bacteria in milk during storage. Therefore, the thermoresistant psychrotrophic bacterial protease(s) tested in this study may affect the manufacture or quality of Cheddar cheese during ripening to a relatively limited extent. However, controlling initial levels of proteolytic bacteria in raw milk remains essential, because proteolysis affects the development of flavor and texture in cheese.

Effect of Diet on the Vitamin B Profile of Bovine Milk-Based Protein Ingredients.

The influence of diet on the water-soluble vitamin composition of skim milk powder and whey protein ingredients produced from the milk of cows fed pasture or concentrate-based diets was examined. Fifty-one Holstein-Friesian cows were randomly assigned into three diets (n=17) consisting of outdoor grazing of perennial ryegrass (GRS), perennial ryegrass/white clover (CLV), or indoor feeding of total mixed ration (TMR) for an entire lactation. Raw mid-lactation milk from each group was processed into skim milk powder and further processed to yield micellar casein whey and acid whey. Sweet whey was also produced by renneting of pasteurised whole milk from each system. The water-soluble vitamin profile of each sample was analysed using a combination of direct injection mass spectrometry and reverse-phase liquid chromatography-mass spectrometry. Vitamin B3 and B3-amide concentrations were significantly higher (p < 0.05) in TMR-derived samples than in those from CLV and GRS, respectively. Vitamin B1, B2, and B7 concentrations were significantly higher in GRS and CLV-derived samples than those from TMR. Significant differences in vitamins B1, B2, and B3-amide were also observed between protein ingredient types. This study indicates that bovine feeding systems have a significant effect on B vitamin composition across a range of protein ingredient types.

Dietary Compounds Influencing the Sensorial, Volatile and Phytochemical Properties of Bovine Milk.

The main aim of this study was to evaluate the volatile profile, sensory perception, and phytochemical content of bovine milk produced from cows fed on three distinct feeding systems, namely grass (GRS), grass/clover (CLV), and total mixed ration (TMR). Previous studies have identified that feed type can influence the sensory perception of milk directly via the transfer of volatile aromatic compounds, or indirectly by the transfer of non-volatile substrates that act as precursors for volatile compounds. In the present study, significant differences were observed in the phytochemical profile of the different feed and milk samples. The isoflavone formonoetin was significantly higher in CLV feed samples, but higher in raw GRS milk, while other smaller isoflavones, such as daidzein, genistein, and apigenin were highly correlated to raw CLV milk. This suggests that changes in isoflavone content and concentration in milk relate to diet, but also to metabolism in the rumen. This study also found unique potential volatile biomarkers in milk (dimethyl sulfone) related to feeding systems, or significant differences in the concentration of others (toluene, p-cresol, ethyl and methyl esters) based on feeding systems. TMR milk scored significantly higher for hay-like flavor and white color, while GRS and CLV milk scored significantly higher for a creamy color. Milk samples were easily distinguishable by their volatile profile based on feeding system, storage time, and pasteurization.

Impact of Bovine Diet on Metabolomic Profile of Skim Milk and Whey Protein Ingredients.

The influence of bovine diet on the metabolome of reconstituted skim milk powder (SMP) and protein ingredients produced from the milk of cows fed on pasture or concentrate-based diets was investigated. Cows were randomly assigned to diets consisting of perennial ryegrass only (GRS), perennial ryegrass/white clover sward (CLV), or indoor total mixed ration (TMR) for an entire lactation. Raw milk obtained from each group was processed at pilot scale, to produce SMP and sweet whey, and SMP was further processed at laboratory scale, to yield ideal whey and acid whey. The total amino acid composition and metabolome of each sample were analyzed, using high-performance cation exchange and a targeted combination of direct-injection mass spectrometry and reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. The nitrogen composition of the products from each of the diets was similar, with one exception being the significantly higher nonprotein nitrogen content in TMR-derived skim milk powder than that from the GRS system. Total amino acid analysis showed significantly higher concentrations of glycine in GRS- and CLV-derived sweet whey and acid whey than in those from TMR. The cysteine contents of CLV-derived ideal whey and acid whey were significantly higher than for TMR, while the valine content of GRS-derived acid whey was significantly higher than TMR. The phenylalanine content of GRS-derived ideal whey was significantly higher than that from CLV. Metabolomic analysis showed significantly higher concentrations of the metabolites glutamine, valine, and phosphocreatine in each ingredient type derived from TMR than those from GRS or CLV, while the serine content of each GRS-derived ingredient type was significantly higher than that in TMR-derived ingredients. These results demonstrate that the type of bovine feeding system used can have a significant effect on the amino acid composition and metabolome of skim milk and whey powders and may aid in the selection of raw materials for product manufacture, while the clear separation between the samples gives further evidence for distinguishing milk products produced from different feeding systems based on LC-MS/MS.

The "Grass-Fed" Milk Story: Understanding the Impact of Pasture Feeding on the Composition and Quality of Bovine Milk.

Milk is a highly nutritious food that contains an array of macro and micro components, scientifically proven to be beneficial to human health. While the composition of milk is influenced by a variety of factors, such as genetics, health, lactation stage etc., the animal's diet remains a key mechanism by which its nutrition and processing characteristics can be altered. Pasture feeding has been demonstrated to have a positive impact on the nutrient profile of milk, increasing the content of some beneficial nutrients such as Omega-3 polyunsaturated fatty acids, vaccenic acid, and conjugated linoleic acid (CLA), while reducing the levels of Omega-6 fatty acids and palmitic acid. These resultant alterations to the nutritional profile of "Grass-Fed" milk resonate with consumers that desire healthy, "natural", and sustainable dairy products. This review provides a comprehensive comparison of the impact that pasture and non-pasture feeding systems have on bovine milk composition from a nutritional and functional (processability) perspective, highlighting factors that will be of interest to dairy farmers, processors, and consumers.

Physicochemical properties of whole milk powder derived from cows fed pasture or total mixed ration diets.

This study examined the effect of dietary factors on compositional and functional properties of whole milk powder (WMP) produced from bovine milk. Raw milk samples were obtained from 3 groups of 18 Holstein Friesian spring-calving cows randomly assigned to diets based on perennial ryegrass (GRS), perennial ryegrass/white clover sward (CLV), and total mixed ration (TMR). Raw milks obtained in late lactation were subsequently standardized for fat, heat-treated (90°C for 30 s), evaporated, and homogenized before spray drying. The WMP produced from each diet were analyzed to determine differences in color, particle size distribution, heat coagulation time, yogurt gelation, texture profile, and protein profile due to each diet. Significant differences in heat coagulation time were observed between the CLV and TMR samples, whereas color values were significantly different between GRS and TMR samples. No significant differences in gross composition, protein profile, or whey protein nitrogen index were found between the 3 WMP samples. Average D90 values (the particle size at which 90% of the particles were smaller than the specified size) for fat globules were significantly lower in the TMR sample compared with the GRS and CLV samples. Yogurts produced from GRS- and CLV-derived WMP had significantly higher elastic moduli (G') than those produced from TMR-derived WMP. Similarly, texture profile analysis revealed significantly higher firmness values in yogurt samples derived from CLV compared with TMR samples. Our data characterize the effect of these diets on the composition and functional properties of fat-standardized WMP, suggesting better yogurt functionality and thermal stability in WMP derived from pasture-based bovine diets.

Influence of Supplemental Feed Choice for Pasture-Based Cows on the Fatty Acid and Volatile Profile of Milk.

The purpose of this study was to examine the impact of a variety of supplemental feeds on the composition and quality of milk in a pasture-based dairy system. Four pasture-supplemented feeding systems were compared: Group 1 supplementation with 16% crude protein parlour concentrate (CONC); Group 2 supplementation with palm kernel expeller plus parlour concentrate (PKE); Group 3 supplemented with soya hulls plus parlour concentrate (SOYA); Group 4 was supplemented with molassed beet pulp plus parlour concentrate (BEET). Supplemental feeding system was demonstrated to have a significant effect on the size of native casein micelles and the gelation properties of milks. While CONC feeding produced significantly higher casein micelle size, gel strength (Young's Modulus) was significantly negatively correlated with casein micelle size. Supplemental feeding system had a significant effect on a number of fatty acids (FA) and indices derived therefrom, including total saturated and unsaturated fatty acids, de novo produced FA, omega 3, and omega 6 FA. The volatile profile of milks was also affected by supplemental feed choice, whereby multivariate analysis demonstrated that the CONC diet was distinctly different to that of the PALM, SOYA, and BEET milks. Multivariate analysis demonstrated that it is possible to distinguish milks from different pasture-supplemented feeding systems by their FA profile.

Pasture Feeding Changes the Bovine Rumen and Milk Metabolome.

The purpose of this study was to examine the effects of two pasture feeding systems-perennial ryegrass (GRS) and perennial ryegrass and white clover (CLV)-and an indoor total mixed ration (TMR) system on the (a) rumen microbiome; (b) rumen fluid and milk metabolome; and (c) to assess the potential to distinguish milk from different feeding systems by their respective metabolomes. Rumen fluid was collected from nine rumen cannulated cows under the different feeding systems in early, mid and late lactation, and raw milk samples were collected from ten non-cannulated cows in mid-lactation from each of the feeding systems. The microbiota present in rumen liquid and solid portions were analysed using 16S rRNA gene sequencing, while ¹H-NMR untargeted metabolomic analysis was performed on rumen fluid and raw milk samples. The rumen microbiota composition was not found to be significantly altered by any feeding system in this study, likely as a result of a shortened adaptation period (two weeks' exposure time). In contrast, feeding system had a significant effect on both the rumen and milk metabolome. Increased concentrations of volatile fatty acids including acetic acid, an important source of energy for the cow, were detected in the rumen of TMR and CLV-fed cows. Pasture feeding resulted in significantly higher concentrations of isoacids in the rumen. The ruminal fluids of both CLV and GRS-fed cows were found to have increased concentrations of p-cresol, a product of microbiome metabolism. CLV feeding resulted in increased rumen concentrations of formate, a substrate compound for methanogenesis. The TMR feeding resulted in significantly higher rumen choline content, which contributes to animal health and milk production, and succinate, a product of carbohydrate metabolism. Milk and rumen-fluids were shown to have varying levels of dimethyl sulfone in each feeding system, which was found to be an important compound for distinguishing between the diets. CLV feeding resulted in increased concentrations of milk urea. Milk from pasture-based feeding systems was shown to have significantly higher concentrations of hippuric acid, a potential biomarker of pasture-derived milk. This study has demonstrated that ¹H-NMR metabolomics coupled with multivariate analysis is capable of distinguishing both rumen-fluid and milk derived from cows on different feeding systems, specifically between indoor TMR and pasture-based diets used in this study.