Quantitative Bottom-Up Glycomic Analysis of Polysaccharides in Food Matrices Using Liquid Chromatography-Tandem Mass Spectrometry.

Carbohydrates are the most abundant biomolecules in nature, and specifically, polysaccharides are present in almost all plants and fungi. Due to their compositional diversity, polysaccharide analysis remains challenging. Compared to other biomolecules, high-throughput analysis for carbohydrates has yet to be developed. To address this gap in analytical science, we have developed a multiplexed, high-throughput, and quantitative approach for polysaccharide analysis in foods. Specifically, polysaccharides were depolymerized using a nonenzymatic chemical digestion process followed by oligosaccharide fingerprinting using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). Both label-free relative quantitation and absolute quantitation were done based on the abundances of oligosaccharides produced. Method validation included evaluating recovery for a range of polysaccharide standards and a breakfast cereal standard reference material. Nine polysaccharides (starch, cellulose, ß-glucan, mannan, galactan, arabinan, xylan, xyloglucan, chitin) were successfully quantitated with sufficient accuracy (5-25% bias) and high reproducibility (2-15% CV). Additionally, the method was used to identify and quantitate polysaccharides from a diverse sample set of food samples. Absolute concentrations of nine polysaccharides from apples and onions were obtained using an external calibration curve, where varietal differences were observed in some of the samples. The methodology developed in this study will provide complementary polysaccharide-level information to deepen our understanding of the interactions of dietary polysaccharides, gut microbial community, and human health.

Individual Variabilities in Adipose Stem Cell Proliferation, Gene Expression and Responses to Lipopolysaccharide Stimulation.

Adipose stem cells (ASCs) are reported to play a role in normal physiology as well as in inflammation and disease. The objective of this work was to elucidate inter-individual differences in growth, gene expression and response to inflammatory stimuli in ASCs from different donors. Human ASC1 (male donor) and ASC2 (female donor) were purchased from Lonza (Walkersville, MD). Cell proliferation was determined by the sulforhodamine B assay. After time-dependent treatment of ASCs with or without bacterial lipopolysaccharide (LPS), marker gene mRNAs for proliferation, steroid hormones, and xenobiotic and immune pathways were determined using RT-PCR, and secreted cytokine levels in media were measured using the Bio-Plex cytokine assay kit. ASCs from both donors expressed androgen receptors but not estrogen receptors. ASC2 had a 2-fold higher proliferation rate and a 6-fold higher level of proliferation marker Ki67 mRNA than ASC1. ASC2 exhibited significantly greater fold induction of TNF-α and CCL2 by LPS compared to ASC1. TNF-α and GM-CSF protein levels were also significantly higher in the LPS-induced ASC2 media, but IL-6 secretion was higher in the LPS-induced ASC1 media. Our findings suggest that inter-individual variability and/or possible sex differences exist in ASCs, which may serve as a key determinant to inflammatory responses of ASCs.

Improving natural product research translation: From source to clinical trial.

While great interest in health effects of natural product (NP) including dietary supplements and foods persists, promising preclinical NP research is not consistently translating into actionable clinical trial (CT) outcomes. Generally considered the gold standard for assessing safety and efficacy, CTs, especially phase III CTs, are costly and require rigorous planning to optimize the value of the information obtained. More effective bridging from NP research to CT was the goal of a September, 2018 transdisciplinary workshop. Participants emphasized that replicability and likelihood of successful translation depend on rigor in experimental design, interpretation, and reporting across the continuum of NP research. Discussions spanned good practices for NP characterization and quality control; use and interpretation of models (computational through in vivo) with strong clinical predictive validity; controls for experimental artefacts, especially for in vitro interrogation of bioactivity and mechanisms of action; rigorous assessment and interpretation of prior research; transparency in all reporting; and prioritization of research questions. Natural product clinical trials prioritized based on rigorous, convergent supporting data and current public health needs are most likely to be informative and ultimately affect public health. Thoughtful, coordinated implementation of these practices should enhance the knowledge gained from future NP research.

Dose-Dependent Responses of I3C and DIM on T-Cell Activation in the Human T Lymphocyte Jurkat Cell Line.

Indole-3-carbinol (I3C) and its dimer diindolylmethane (DIM) are bioactive metabolites of a glucosinolate, glucobrassicin, found in cruciferous vegetables. Both I3C and DIM have been reported to possess pro-apoptotic, anti-proliferative and anti-carcinogenic properties via modulation of immune pathways. However, results from these studies remain inconclusive since they lack thorough evaluation of these bioactives' physiological versus pharmacological effects. In the present study, we investigated I3C and DIM's dose-dependent effects on cytokines production in human T lymphocytes Jurkat cell line (Clone E6-1). The results showed that I3C and DIM pretreatment, at higher concentrations of 50 and 10 µM, respectively, significantly increased PMA/ionomycin-induced interleukin-2 (IL-2), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) production, measured by real time polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). As a plausible mechanism underlying such pronounced cytokine release, we found robust increase in downstream nuclear factor κB (NF-κB) and nuclear factor of activated T-cells 1 (NFAT1) signaling with I3C pretreatment, whereas DIM pretreatment only significantly induced NF-κB activation, but not NFAT1. We hypothesize that I3C/DIM pretreatment primes the T cells to become hyperresponsive upon PMA/ionomycin stimulation which in turn differentially induces two major downstream Ca2+-dependent inflammatory pathways, NF-κB and NFAT1. Our data show novel insights into the mechanisms underlying induction of pro-inflammatory cytokine release by pharmacological concentrations of I3C and DIM, an effect negligible under physiological conditions.

Increased palmitate intake: higher acylcarnitine concentrations without impaired progression of ß-oxidation.

Palmitic acid (PA) is associated with higher blood concentrations of medium-chain acylcarnitines (MCACs), and we hypothesized that PA may inhibit progression of FA ß-oxidation. Using a cross-over design, 17 adults were fed high PA (HPA) and low PA/high oleic acid (HOA) diets, each for 3 weeks. The [1-(13)C]PA and [13-(13)C]PA tracers were administered with food in random order with each diet, and we assessed PA oxidation (PA OX) and serum AC concentration to determine whether a higher PA intake promoted incomplete PA OX. Dietary PA was completely oxidized during the HOA diet, but only about 40% was oxidized during the HPA diet. The [13-(13)C]PA/[1-(13)C]PA ratio of PA OX had an approximate value of 1.0 for either diet, but the ratio of the serum concentrations of MCACs to long-chain ACs (LCACs) was significantly higher during the HPA diet. Thus, direct measurement of PA OX did not confirm that the HPA diet caused incomplete PA OX, despite the modest, but statistically significant, increase in the ratio of MCACs to LCACs in blood.

Sex-specific metabolic adaptations from in utero exposure to particulate matter derived from combustion of petrodiesel and biodiesel fuels.

Maternal exposure to particulate matter derived from diesel exhaust has been shown to cause metabolic dysregulation, neurological problems, and increased susceptibility to diabetes in the offspring. Diesel exhaust is a major source of air pollution and the use of biodiesel (BD) and its blends have been progressively increasing throughout the world; however, studies on the health impact of BD vs. petrodiesel combustion-generated exhaust have been controversial in part, due to differences in the chemical and physical nature of the associated particulate matter (PM). To explore the long-term impact of prenatal exposure, pregnant mice were exposed to PM generated by combustion of petrodiesel (B0) and a 20% soy BD blend (B20) by intratracheal instillation during embryonic days 9-17 and allowed to deliver. Offspring were then followed for 52 weeks. We found that mother's exposure to B0 and B20 PM manifested in striking sex-specific phenotypes with respect to metabolic adaptation, maintenance of glucose homeostasis, and medial hypothalamic glial cell makeup in the offspring. The data suggest PM exposure limited to a narrower critical developmental window may be compensated for by the mother and/or the fetus by altered metabolic programming in a marked sex-specific and fuel-derived PM-specific manner, leading to sex-specific risk for diseases related to environmental exposure later in life.

Nutrients and non-essential metals in darkibor kale grown at urban and rural farms: A pilot study.

Kale is a nutrient-dense leafy vegetable associated with wide-ranging health benefits. It is tolerant of drought and temperature fluctuations, and could thus serve an increasingly important role in providing a safe and nutritious food supply during the climate crisis, while kale's ease of cultivation and ability to be grown in a wide range of soils make it a good fit for urban agriculture. In this pilot study we explored potential differences between kale grown at urban versus rural farms. We planted kale seedlings (Darkibor variety) at three urban and four rural farms in and around Baltimore City, Maryland, instructed farmers to cultivate them using their usual growing practices, harvested the kale from fields and points of distribution, and analyzed it for concentrations of carotenoids, vitamins C and K1, ten nutritional elements, and eight non-essential metals. Although sample sizes for some analyses were in some cases too small to produce statistically significant results, we identified potentially meaningful differences in concentrations of several components between urban and rural kale samples. Compared to urban samples, mean concentrations of carotenoids and vitamins were 22-38% higher in rural field samples. By contrast, mean concentrations for eight nutritional elements were higher in urban field samples by as much as 413% for iron. Compared to rural field samples, mean concentrations of nine non-essential metals were higher in urban samples, although lead and cadmium concentrations for all samples were below public health guidelines. Some urban-rural differences were more pronounced than those identified in prior research. For six elements, variance within urban and rural farms was greater than variance between urban and rural farms, suggesting urbanicity may not be the primary driver of some observed differences. For some nutrients, mean concentrations were higher than upper ranges reported in prior estimates, suggesting kale may have the potential to be more nutrient-dense than previously estimated. The nutritive and metals composition of this important crop, and the factors that influence it, merit continued investigation given its growing popularity.

A multi-glycomic platform for the analysis of food carbohydrates.

Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan-microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health.

Protein and amino acid supplementation in older humans.

The aging process is a continuum throughout life and often associated with deterioration of body function as well as accumulation of chronic disabilities and of disease. The impact of nutritional status on morbidity and mortality is unquestioned. Malnutrition increases the risk for frailty and nutritional deficits can influence immune status, response to medical treatments and recovery from acute illnesses, including surgery. Health-promoting interventions implemented individually, such as exercise programs, preventive home visits, comprehensive geriatric evaluation and management, and attention to adequate nutrition with or without nutritional supplements, have been shown in separate studies to be both feasible and effective in reducing age-related deterioration. Protein and its constituent amino acids (AA) are key components of any healthy diet. Sarcopenia, the slow but progressive loss of lean muscle mass associated with advancing age, has been the focus of many studies but there is no clear-cut answer to the question of how to restrain the process. The more general question of how the requirements for protein and specific AA change with age continues to be investigated. A shift towards studying the efficacy and safety of specific AA or combination of AA that may sustain and/or enhance physiologic processes, ranging from specific tissue metabolism to overall function (e.g. exercise performance, immune function, cognition, and chronic disease development) has occurred. This review focuses on recent studies examining the use of specific AA or mixtures as supplements in the elderly and whether/how AA may assist in the maintenance of health and independence.

Soy biodiesel and petrodiesel emissions differ in size, chemical composition and stimulation of inflammatory responses in cells and animals.

Debate about the biological effects of biodiesel exhaust emissions exists due to variation in methods of exhaust generation and biological models used to assess responses. Because studies in cells do not necessarily reflect the integrated response of a whole animal, experiments were conducted in two human cell lines representing bronchial epithelial cells and macrophages and female mice using identical particle suspensions of raw exhaust generated by a Volkswagen light-duty diesel engine using petrodiesel (B0) and a biodiesel blend (B20: 20% soy biodiesel/80% B0 by volume). Tailpipe particle emissions measurement showed B0 generated two times more particle mass, larger ultrafine particle number distribution modes, and particles of more nonpolar organic composition than the B20 fuel. Biological assays (inflammatory mediators, oxidative stress biomarkers) demonstrated that particulate matter (PM) generated by combustion of the two fuels induced different responses in in vitro and in vivo models. Concentrations of inflammatory mediators (Interleukin-6, IL-6; Interferon-gamma-induced Protein 10, IP-10; Granulocyte-stimulating factor, G-CSF) in the medium of B20-treated cells and in bronchoalveolar lavage fluid of mice exposed to B20 were ∼20-30% higher than control or B0 PM, suggesting that addition of biodiesel to diesel fuels will reduce PM emissions but not necessarily adverse health outcomes.

Comprehensive oligosaccharide profiling of commercial almond milk, soy milk, and soy flour.

Oligosaccharides are known for several bioactivities on health, however, in sensitive individuals, can cause intestinal discomfort. This study aimed to investigate the oligosaccharide profiles in selected plant-based food products. A quantification method based on high-performance anion-exchange chromatography-pulsed amperometric detection was developed, validated, and used to measure major oligosaccharides. Additional low-abundant oligosaccharides and glycosides were characterized by liquid chromatography-tandem mass spectrometry and glycosidases. The summed concentration of raffinose, stachyose, and verbascose ranged from 0.12-0.19 mg/g in almond milk, 3.6-6.4 mg/g in soy milk, and 74-77 and 4.8-57 mg/g in defatted and full-fat soy four. Over 80 different oligosaccharides were characterized. Novel compounds, 2,3-butanediol glycosides, were identified in almond milk. Low-abundant oligosaccharides represented 25 %, 6 %, and 10 % of total OS in almond milk, soy milk, and soy flour, respectively. The data here are useful to estimate oligosaccharide consumption from dietary intake and facilitate further studies on their bioactivity.

The National Clinical Care Commission Report to Congress: Leveraging Federal Policies and Programs for Population-Level Diabetes Prevention and Control: Recommendations From the National Clinical Care Commission.

The etiology of type 2 diabetes is rooted in a myriad of factors and exposures at individual, community, and societal levels, many of which also affect the control of type 1 and type 2 diabetes. Not only do such factors impact risk and treatment at the time of diagnosis but they also can accumulate biologically from preconception, in utero, and across the life course. These factors include inadequate nutritional quality, poor access to physical activity resources, chronic stress (e.g., adverse childhood experiences, racism, and poverty), and exposures to environmental toxins. The National Clinical Care Commission (NCCC) concluded that the diabetes epidemic cannot be treated solely as a biomedical problem but must also be treated as a societal problem that requires an all-of-government approach. The NCCC determined that it is critical to design, leverage, and coordinate federal policies and programs to foster social and environmental conditions that facilitate the prevention and treatment of diabetes. This article reviews the rationale, scientific evidence base, and content of the NCCC's population-wide recommendations that address food systems; consumption of water over sugar-sweetened beverages; food and beverage labeling; marketing and advertising; workplace, ambient, and built environments; and research. Recommendations relate to specific federal policies, programs, agencies, and departments, including the U.S. Department of Agriculture, the Food and Drug Administration, the Federal Trade Commission, the Department of Housing and Urban Development, the Environmental Protection Agency, and others. These population-level recommendations are transformative. By recommending health-in-all-policies and an equity-based approach to governance, the NCCC Report to Congress has the potential to contribute to meaningful change across the diabetes continuum and beyond. Adopting these recommendations could significantly reduce diabetes incidence, complications, costs, and inequities. Substantial political resolve will be needed to translate recommendations into policy. Engagement by diverse members of the diabetes stakeholder community will be critical to such efforts.

Associations among Milk Microbiota, Milk Fatty Acids, Milk Glycans, and Inflammation from Lactating Holstein Cows.

Milk oligosaccharides (MOs) can be prebiotic and antiadhesive, while fatty acids (MFAs) can be antimicrobial. Both have been associated with milk microbes or mammary gland inflammation in humans. Relationships between these milk components and milk microbes or inflammation have not been determined for cows and could help elucidate a novel approach for the dairy industry to promote desired milk microbial composition for improvement of milk quality and reduction of milk waste. We aimed to determine relationships among milk microbiota, MFAs, MOs, lactose, and somatic cell counts (SCC) from Holstein cows, using our previously published data. Raw milk samples were collected at three time points, ranging from early to late lactation. Data were analyzed using linear mixed-effects modeling and repeated-measures correlation. Unsaturated MFA and short-chain MFA had mostly negative relationships with potentially pathogenic genera, including Corynebacterium, Pseudomonas, and an unknown Enterobacteriaceae genus but numerous positive relationships with symbionts Bifidobacterium and Bacteroides. Conversely, many MOs were positively correlated with potentially pathogenic genera (e.g., Corynebacterium, Enterococcus, and Pseudomonas), and numerous MOs were negatively correlated with the symbiont Bifidobacterium. The neutral, nonfucosylated MO composed of eight hexoses had a positive relationship with SCC, while lactose had a negative relationship with SCC. One interpretation of these trends might be that in milk, MFAs disrupt primarily pathogenic bacterial cells, causing a relative increase in abundance of beneficial microbial taxa, while MOs respond to and act on pathogenic taxa primarily through antiadhesive methods. Further research is needed to confirm the potential mechanisms driving these correlations. IMPORTANCE Bovine milk can harbor microbes that cause mastitis, milk spoilage, and foodborne illness. Fatty acids found in milk can be antimicrobial and milk oligosaccharides can have antiadhesive, prebiotic, and immune-modulatory effects. Relationships among milk microbes, fatty acids, oligosaccharides, and inflammation have been reported for humans. To our knowledge, associations among the milk microbial composition, fatty acids, oligosaccharides, and lactose have not been reported for healthy lactating cows. Identifying these potential relationships in bovine milk will inform future efforts to characterize direct and indirect interactions of the milk components with the milk microbiota. Since many milk components are associated with herd management practices, determining if these milk components impact milk microbes may provide valuable information for dairy cow management and breeding practices aimed at minimizing harmful and spoilage-causing microbes in raw milk.

Perspective: A Research Roadmap about Ultra-Processed Foods and Human Health for the United States Food System: Proceedings from an Interdisciplinary, Multi-Stakeholder Workshop.

Our objective was to convene interdisciplinary experts from government, academia, and industry to develop a Research Roadmap to identify research priorities about processed food intake and risk for obesity and cardiometabolic diseases (CMD) among United States populations. We convened attendees at various career stages with diverse viewpoints in the field. We held a "Food Processing Primer" to build foundational knowledge of how and why foods are processed, followed by presentations about how processed foods may affect energy intake, obesity, and CMD risk. Breakout groups discussed potential mechanistic and confounding explanations for associations between processed foods and obesity and CMD risk. Facilitators created research questions (RQs) based on key themes from discussions. Different breakout groups convened to discuss what is known and unknown for each RQ and to develop sub-RQs to address gaps. Workshop attendees focused on ultra-processed foods (UPFs; Nova Group 4) because the preponderance of evidence is based on this classification system. Yet, heterogeneity and subjectivity in UPF classification was a challenge for RQ development. The 6 RQs were: 1) What objective methods or measures could further categorize UPFs, considering food processing, formulation, and the interaction of the two? 2) How can exposure assessment of UPF intake be improved? 3) Does UPF intake influence risk for obesity or CMDs, independent of diet quality? 4) What, if any, attributes of UPFs influence ingestive behavior and contribute to excess energy intake? 5) What, if any, attributes of UPFs contribute to clinically meaningful metabolic responses? 6) What, if any, external environmental factors lead people to consume high amounts of UPFs? Uncertainty and complexity around UPF intake warrant further complementary and interdisciplinary causal, mechanistic, and methodological research related to obesity and CMD risk to understand the utility of applying classification by degree of processing to foods in the United States.

Differential activation of the inflammasome in THP-1 cells exposed to chrysotile asbestos and Libby "six-mix" amphiboles and subsequent activation of BEAS-2B cells.

Inflammatory responses of THP-1 cells (macrophage cell line) exposed to chrysotile asbestos (Chry) and Libby six-mix (LIB) and the subsequent impact on bronchial epithelial cells were determined. Direct treatment of THP-1 cells with Chry caused cell death, activation of caspase-1 and release of IL-1ß, while the addition of caspase-1 inhibitor, Z-YVAD-FMK, reduced IL-1ß, suggesting that Chry activated the caspase-1 mediated Nod-like receptor protein 3 (NLRP3) inflammasome; by comparison, LIB had less effects on all of these parameters. Expression of antioxidant enzymes, protein oxidation and nitration, and lipid peroxides in THP-1 cells treated with the two particles suggest that LIB generated more reactive oxygen species (ROS) than the same dose of Chry. Differences in fiber length and surface area suggest a possible role for particulate size in the differential activation of the inflammasome. BEAS-2B cells, representing the bronchial epithelium, treated with supernatants of medium from Chry- or LIB-treated THP-1 cells (conditioned medium) activated the MAPK cascade, increased phosphorylation of ERK and Cot (MAP3K8), increased AP-1 binding activity and induced IL-6 release. To verify that IL-1ß from THP-1 cells was responsible for activation of BEAS-2B, conditioned medium with added IL-1Ra, an IL-1ß antagonist, was applied to BEAS-2B. Results show that IL-1Ra attenuated effects of conditioned medium, supporting a role of IL-1ß, as a secondary mediator, in the transduction of inflammatory signaling from the macrophage to epithelial cells. The effects of LIB-conditioned medium appeared to be less dependent on IL-1ß. In conclusion, Chry and LIB induce differential inflammatory responses in THP-1 cells that subsequently lead to differential effects in epithelial cells.

USDA's FoodData Central: what is it and why is it needed today?

FoodData Central (FDC) is the center of the USDA-based food-composition information web. It is an integrated data system that presently provides-in 1 place-5 distinct types of data containing information on food and nutrient profiles. Each data type has a unique purpose. Two of the data types-Foundation Foods (FF) and Experimental Foods (EF)-represent "a bridge to the future" in food and nutrient composition. They provide data and metadata that have never previously been available from a database. The other 3 data types are well established and familiar to many users: Standard Reference (SR) Legacy, Food and Nutrient Database for Dietary Studies (FNDDS), and Global Branded Foods Products Database (GBFPD). After >100 y of maintaining food-composition data within the USDA, it was clear that change was needed to respond to the rapid increase in the number and variety of foods in the food supply, evolution of analytical approaches, and new agricultural practices and products. FDC is USDA's answer to the challenge of providing reliable, web-based, transparent, and easily accessible information about the nutrients and other components of foods to meet the increasingly diverse needs of many audiences, including public health professionals, agricultural and environmental researchers, policy makers, nutrition professionals, health care providers, product developers, and the public at large.

Dietary Fiber to Starch Ratio Affects Bovine Milk Oligosaccharide Profiles.

Background: Bovine milk oligosaccharides (BMOs) have several demonstrated and hypothesized benefits including roles in cognitive development and antipathogenic activities, making them promising ingredients for infant formulas and nutraceutical applications. BMO extraction from bovine milk is challenged by low concentrations relative to nonbioactive simple sugars like lactose. BMO abundances are known to vary with a cow's lactation stage, breed, and parity, but these characteristics are difficult to modify in existing dairy herds. In contrast, diet modification is an accessible target, and is already known to influence milk yield, lipid content, protein levels, and monosaccharide compositions. Objectives: To determine the impact of a low starch high fiber versus a high starch low fiber diet on overall BMO profiles and individual BMO abundances in Holstein dairy cattle. Methods: Milk samples were collected from 59 midlactation Holsteins in a crossover study featuring dietary modification with either a low starch high fiber or high starch low fiber feed. BMO profiles were evaluated by nano-LC quadrupole time-of-flight tandem MS, and differences in BMO abundances between diets were evaluated using linear mixed effects modeling. Results: A total of 19 BMOs were identified across the sample set, including 4 large fucosylated compounds. Seven BMOs were found to have significantly more positive percent changes in yield-adjusted abundance from the pre-experiment baseline period for milk samples collected during feeding with the low starch high fiber diet compared with the high starch low fiber diet. Conclusions: Consuming the low starch high fiber diet promoted greater overall BMO production than the high starch low fiber diet in a population of midlactation Holsteins. Additionally, this study afforded the opportunity to investigate the impact of other factors potentially influencing BMO abundances, furthering understanding of how dairy herd management practices can positively impact milk composition and support the potential use of BMOs as functional ingredients.

A Low-Starch and High-Fiber Diet Intervention Impacts the Microbial Community of Raw Bovine Milk.

Background: A more sustainable dairy cow diet was designed that minimizes use of feed components digestible by monogastric animals by increasing the quantity of forages. Objectives: This study determined if feeding lactating cows the more sustainable, low-starch and high-fiber (LSHF) diet was associated with changes in raw milk microbiota composition and somatic cell count (SCC). Methods: In a crossover design, 76 lactating Holstein cows were assigned to an LSHF diet or a high-starch and low-fiber (HSLF) diet, similar to common dairy cow diets in the United States, for 10 wk then placed on the opposite diet for 10 wk. The LSHF diet contained greater quantities of forages, beet pulp, and corn distillers' grain, but contained less canola meal and no high-moisture corn compared with the HSLF diet. Raw milk samples were collected from each cow 4-5 d before intervention and 5 wk into each diet treatment. Within 4 d, additional milk samples were collected for measurement of SCC using Fossmatic 7. The microbial community was determined by sequencing the 16S rRNA gene V4-V5 region and analyzing sequences with QIIME2. After quality filtering, 53 cows remained. Results: Raw milk microbial communities differed by diet and time. Taxa associated with fiber consumption, such as Lachnospiraceae, Lactobacillus, Bacteroides, and Methanobrevibacter, were enriched with the LSHF diet. Meanwhile, taxa associated with mastitis, such as Pseudomonas, Stenotrophomonas, and Enterobacteriaceae, were enriched with the HSLF diet. Relatedly, an interaction of diet and time was found to impact SCC. Conclusions: In raw milk, consumption of an LSHF diet compared with an HSLF diet was associated with changes in abundance of microbes previously associated with fiber consumption, udder health, and milk spoilage. Further research is needed to determine if an LSHF diet indeed leads to lower rates of mastitis and milk spoilage, which could benefit the dairy industry.

Identification of High and Low Branched-Chain Fatty Acid-Producing Phenotypes in Holstein Cows following High-Forage and Low-Forage Diets in a Crossover Designed Trial.

BACKGROUND: Branched-chain fatty acids (BCFAs) are rumen-derived fatty acids comprising ∼2% of bovine-milk fatty acids. BCFAs possess anti-inflammatory properties and enriching the BCFA content of bovine milk may provide human health benefits. OBJECTIVE: We determined whether forage content impacts the BCFA content of milk from Holstein cows and identified fatty acid phenotypes in high vs. low BCFA-containing milks. METHODS: Holstein cows (n = 62), fed for 67 d in a crossover design, consumed a diet with high forage and low concentrate (HF:C) and a diet with low forage and high concentrate (LF:C). Milk samples were collected at the end of each treatment period and fatty acid content determined. Paired t-tests, 1-factor ANOVA, sparse partial least-squares discriminant analysis (sPLSDA), and Pearson's correlation analysis were used to analyze the data. RESULTS: The total milk fatty acid concentration for cows fed the HF:C diet was greater than that of cows fed the LF:C diet (4.2 ± 0.7 g/100 mL vs. 3.9 ± 0.9 g/100 mL). sPLSDA demonstrated separation of the dietary treatments, with BCFAs and odd-chain fatty acids as primary determinants. Total BCFA content in milk fat was elevated by HF:C intake compared with LF:C intake (1.80 vs. 1.68%). Quintile separation of high vs. low BCFA milks resulted in 4 groups: HF:C /low BCFAs, HF:C /high BCFAs; LF:C /low BCFAs, and LF:C /high BCFAs. Milks from the high BCFA quintiles had lower palmitic acid content (29.6% vs. 34.4%) but higher oleic acid content than milks from the low BCFA quintiles (19.7% vs. 17.0%). Some cows were identified as high BCFA producers or low BCFA producers regardless of diet. CONCLUSIONS: BCFA content of milk is diet-sensitive but variation in responses exists. The potential to produce milk with high BCFA content and lower SFA content needs further study.