Milk phospholipids protect Bifidobacterium longum subsp. infantis during in vitro digestion and enhance polysaccharide production.

Bifidobacterium longum subsp. infantis is associated with the gut microbiota of breast-fed infants. Bifidobacterium infantis promotes intestinal barrier and immune function through several proposed mechanisms, including interactions between their surface polysaccharides, the host, and other gut microorganisms. Dairy foods and ingredients are some of the most conspicuous food-based niches for this species and may provide benefits for their delivery and efficacy in the gut. Milk phospholipid (MPL)-rich ingredients have been increasingly recognized for their versatile benefits to health, including interactions with the gut microbiota and intestinal cells. Therefore, our objective was to investigate the capacity for MPL to promote survival of B. infantis during simulated digestion and to modulate bacterial polysaccharide production. To achieve these aims, B. infantis was incubated with or without 0.5% MPL in de Man, Rogosa, and Sharpe (MRS) media at 37°C under anaerobiosis. Survival across the oral, gastric, and intestinal phases using in vitro digestion was measured using plate count, along with adhesion to goblet-like intestinal cells. MPL increased B. infantis survival at the end of the intestinal phase by at least 7% and decreased adhesion to intestinal cells. The bacterial surface characteristics, which may contribute to these effects, were assessed by ζ-potential, changes in surface proteins using comparative proteomics, and production of bound polysaccharides. MPL decreased the surface charge of the bifidobacteria from -17 to -24 mV and increased a 50 kDa protein (3-fold) that appears to be involved in protection from stress. The production of bound polysaccharides was measured using FTIR, HPLC, and TEM imaging. These techniques all suggest an increase in bound polysaccharide production at least 1.7-fold in the presence of MPL. Our results show that MPL treatment increases B. infantis survival during simulated digestion, induces a stress resistance surface protein, and yields greater bound polysaccharide production, suggesting its use as a functional ingredient to enhance probiotic and postbiotic effects.

Partition of milk phospholipids during ice cream manufacturing.

The distribution of phospholipids (PL) within the fat and serum phase of ice cream manufacturing was evaluated through partition coefficients (KPL) after mixing, pasteurization, freezing, and hardening. Ice creams containing about 40.41 ± 3.45 (± standard deviation; control formulation) and 112.29 ± 9.06 (enriched PL formulation) mg of PL per g of fat were formulated with nonfat dry milk and ß-serum, respectively. Overall, the KPL were lower than 1, indicating that the PL were predominantly found in the fat phase, and only a small amount was left in the serum and sediment. Confocal micrographs visually confirmed this generalization. The addition of PL significantly increased the viscosity of the mixes between 4- and 9-fold, depending on the shear rate. Additionally, mixes containing high PL exhibited higher yield stress than those formulated with low PL (0.15 ± 0.09 and 0.016 ± 0.08 Pa, respectively). Ice creams with high PL delayed the onset of meltdown and exhibited a slower rate of a meltdown than low-PL ice creams (18.53 ± 0.57 and 14.83 ± 0.85 min, and 1.01 ± 0.05 and 0.71 ± 0.04% min-1, respectively). This study provides useful guidelines for manufacturing ice cream enriched in milk PL. Additionally, the use of ß-serum, a byproduct stream, as a source of PL is illustrated. The development will require studying the sensorial description of the product as well as consumer acceptance.

Milk-Fat-Globule-Membrane-Enriched Dairy Milk Compared with a Soy-Lecithin-Enriched Beverage Did Not Adversely Affect Endotoxemia or Biomarkers of Gut Barrier Function and Cardiometabolic Risk in Adults with Metabolic Syndrome: A Randomized Controlled Crossover Trial.

Full-fat dairy milk may protect against cardiometabolic disorders, due to the milk fat globule membrane (MFGM), through anti-inflammatory and gut-health-promoting activities. We hypothesized that a MFGM-enriched milk beverage (MEB) would alleviate metabolic endotoxemia in metabolic syndrome (MetS) persons by improving gut barrier function and glucose tolerance. In a randomized crossover trial, MetS persons consumed for two-week period a controlled diet with MEB (2.3 g/d milk phospholipids) or a comparator beverage (COMP) formulated with soy phospholipid and palm/coconut oil. They then provided fasting blood and completed a high-fat/high-carbohydrate test meal challenge for evaluating postprandial metabolism and intestinal permeability. Participants had no adverse effects and achieved high compliance, and there were no between-trial differences in dietary intakes. Compared with COMP, fasting endotoxin, glucose, incretins, and triglyceride were unaffected by MEB. The meal challenge increased postprandial endotoxin, triglyceride, and incretins, but were unaffected by MEB. Insulin sensitivity; fecal calprotectin, myeloperoxidase, and short-chain fatty acids; and small intestinal and colonic permeability were also unaffected by MEB. This short-term study demonstrates that controlled administration of MEB in MetS persons does not affect gut barrier function, glucose tolerance, and other cardiometabolic health biomarkers, which contradicts observational evidence that full-fat milk heightens cardiometabolic risk. Registered at ClinicalTrials.gov (NCT03860584).

Impact of ultra-shear technology on quality attributes of model dairy-pea protein dispersions with different fat levels.

This study investigated the impact of ultra-shear technology (UST) processing on dairy-pea protein dispersions with different fat levels. Raw milk, skim milk, and cream, as well as model dispersions with combinations of dairy products and pea protein (i.e., raw milk with pea protein, skim milk with pea protein, and cream with pea protein) were employed as test samples. UST experiments were conducted at a pressure of 400 MPa and 70 °C shear valve exit temperature. The UST treatment increased the viscosity of the dispersions and the increases depended on the fat level. Dairy-pea protein dispersions from raw milk and skim milk were shear thinning and mathematically described by the power-law model defined by the consistency coefficient, K (Pa·sn) and the flow behavior index, n. UST treated cream + pea protein dispersions produced structures with gel-like characteristics. Microstructure and particle size analysis determined by laser scanning microscope revealed a reduction in particle size after UST treatment in raw milk + pea protein and skim milk + pea protein dispersions up to 7.55 and 8.30 µm, respectively. In contrast, the particle mean diameter of cream + pea protein dispersions increased up to 77.20 µm after the UST treatment. Thus, the effect of UST on the particle size and rheological behavior of the dispersions depended on the fat level. UST-treated dispersions were stable with no visible phase separation or sedimentation upon centrifugation at 4000×g for 30 min (4 °C). Heat treatment and freeze-thaw treatment of UST-treated samples showed stable blends immediately after the treatments, but subsequent centrifugation showed solid separation. Results from the study suggest that UST is a potential technology to produce stable dairy + pea protein liquids foods with different rheological characteristics for diverse applications.

Screening and characterization of ß-galactosidase activity in lactic acid bacteria for the valorization of acid whey.

ß-Galactosidase is an enzyme produced by some strains of lactic acid bacteria (LAB) commonly found in dairy products; however, industrial demand for these enzymes is still low. Acid whey (AW), a lactose-rich byproduct, has large output from cottage cheese and remains unexploited. The purpose of this study was to understand the production mechanism of ß-galactosidase from LAB using AW as a culture medium. First, bioinformatics analysis was conducted on 15 species of LAB. Then, 24 strains were selected and inoculated in de Man, Rogosa, and Sharpe (MRS) broth and in AW medium to compare the bacterial kinetic growth and ß-galactosidase production. Bacterial growth and total protein activity were measured using spectrophotometric techniques. ß-Galactosidase activity was determined by 2 methods: following the hydrolysis of o-nitrophenyl-ß-d-galactopyranoside and of 5-bromo-4-chloro-3-indoyl-ß-d-galactopyranoside (X-gal) in tryptic soy agar plates. The relative expression of the ß-galactosidase gene was performed using real-time quantitative PCR. Despite generally lower growth in AW, 18 strains showed higher ß-galactosidase activity when grown in AW compared with MRS medium. The highest ß-galactosidase activity in AW was in Lactobacillus helveticus strain OSU-PECh-4A, which showed almost 5 times higher activity than average. Analysis of 6 selected strains for expression of the bgal-620 gene found higher overexpression in AW than in MRS, regardless of specific ß-galactosidase activity. Strains of LAB such as OSU-PECh-4A could valorize AW through the production of ß-galactosidase (as an aid to lactose digestion) and production of prebiotic galactooligosaccharides.

Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity.

The leptin receptor (LepR) acts as a signaling nexus for the regulation of glucose uptake and obesity, among other metabolic responses. The functional role of LepR under leptin-deficient conditions remains unclear. This study reports that epiregulin (EREG) governed glucose uptake in vitro and in vivo in Lepob mice by activating LepR under leptin-deficient conditions. Single and long-term treatment with EREG effectively rescued glucose intolerance in comparative insulin and EREG tolerance tests in Lepob mice. The immunoprecipitation study revealed binding between EREG and LepR in adipose tissue of Lepob mice. EREG/LepR regulated glucose uptake without changes in obesity in Lepob mice via mechanisms, including ERK activation and translocation of GLUT4 to the cell surface. EREG-dependent glucose uptake was abolished in Leprdb mice which supports a key role of LepR in this process. In contrast, inhibition of the canonical epidermal growth factor receptor (EGFR) pathway implicated in other EREG responses, increased glucose uptake. Our data provide a basis for understanding glycemic responses of EREG that are dependent on LepR unlike functions mediated by EGFR, including leptin secretion, thermogenesis, pain, growth, and other responses. The computational analysis identified a conserved amino acid sequence, supporting an evolutionary role of EREG as an alternative LepR ligand.

Use of casein micelles to improve the solubility of hydrophobic pea proteins in aqueous solutions via low-temperature homogenization.

The dairy industry struggles to maintain consumer attention in the midst of declining fluid milk sales. Current trends create an opportunity to incorporate plant-based proteins with milk to produce a high-protein, multisourced, functional food product. Plant-based proteins, such as those in peas, can be challenging to use in food systems because of their low solubility and undesirable off-flavors. Casein micelles have unique structural properties that allow for interactions with small ions and larger macromolecules that aid in their noteworthy ability as a nanovehicle for hydrophobic compounds. The objective of this study was to use the inherent structure of the casein micelle along with common dairy processing equipment to create a stable colloidal dispersion of casein micelles with pea protein to improve its solubility in aqueous solutions. We created 3 blends with varying ratios of casein-to-pea protein (90:10, 80:20, 50:50). We subjected the mixtures to 3 cycles of homogenization using a bench-top GEA 2-stage homogenizer at 27,580 kPa maintained at 4°C, followed by pasteurization at 63°C for 30 min. The resulting blends were homogeneous liquids with increased stability due to the lack of protein precipitation. Further protein analysis by HPLC and AA sequencing revealed that vicilin, an insoluble storage protein, was the main pea protein incorporated within the casein micelle structure. These results supported our hypothesis that low-temperature homogenization can successfully be used to create a colloidal dispersion with increased stability, in which insoluble plant-based proteins may be incorporated with casein micelles in an aqueous solution. Additionally, 3-dimensional microscope images of the blends indicated a noticeable difference between the surface roughness upon addition of pea protein to the casein micelle matrix. This research highlights a promising application for other plant-based proteins to be used within the dairy industry to help drive future product innovation while also meeting current processing conditions and consumer demands.

Milk fat globule membrane phospholipids modify adhesion of Lactobacillus to mucus-producing Caco-2/Goblet cells by altering the cell envelope.

The importance of various Lactobacillus strains and milk components, such as the milk fat globule membrane, has been studied from various perspectives and proven to have a positive role in human health. On one end, lactic acid bacteria produce metabolites with direct effect in the immune system, changes of pH in the gut, and antagonistic substances for pathogenic bacteria as well as competition. On the other end, the milk fat globule membrane improves gastrointestinal status by promoting cell proliferation, epithelial tight junction patterns, and development of intestinal epithelial cells. Interaction between beneficial bacteria and milk fat is a natural occurring phenomenon in dairy products; however, it has not been fully characterized. In this work, we studied the effect of milk phospholipids in the adhesion of Lactobacillus to mucus-producing Caco-2/Goblet cell co-cultures and found that treatment with phospholipids produced bacterial cells with increased surface electronegativity, which was correlated with increased bacterial cells adhered to the intestinal model. Moreover, we utilized an original means of characterizing the adhesion using quartz crystal microbalance. All strains studied, experienced modification of adhesion either physicochemical or kinetic parameters studied. Furthermore, by imaging bacterial cells by electron microscopy, we identified that some strains, such as L. acidophillus and L. casei, metabolized MPL, which improved their adhesion to hydrophilic surfaces such as gold. We identified another group of bacteria, such as L. delbrueckii and L. plantarum, that, instead of metabolizing MPL, kept the phospholipids bound irreversibly to the surface of the cell envelope thus decreasing their adherence to gold surfaces. One of the most important aspects of probiotic lactic acid bacteria -besides survival in the stomach-is the colonization and extended resident time in the intestine to effectively change the gut microbiome. We found that bacterial treatment with milk phospholipids enhances adhesion to intestinal models and will in turn, increase the residence time with the concomitant benefits to the consumer.

Improving Human Health with Milk Fat Globule Membrane, Lactic Acid Bacteria, and Bifidobacteria.

The milk fat globule membrane (MFGM), the component that surrounds fat globules in milk, and its constituents have gained significant attention for their gut function, immune-boosting properties, and cognitive-development roles. The MFGM can directly interact with probiotic bacteria, such as bifidobacteria and lactic acid bacteria (LAB), through interactions with bacterial surface proteins. With these interactions in mind, increasing evidence supports a synergistic effect between MFGM and probiotics to benefit human health at all ages. This important synergy affects the survival and adhesion of probiotic bacteria through gastrointestinal transit, mucosal immunity, and neurocognitive behavior in developing infants. In this review, we highlight the current understanding of the co-supplementation of MFGM and probiotics with a specific emphasis on their interactions and colocalization in dairy foods, supporting in vivo and clinical evidence, and current and future potential applications.

Amino Acid Nanofibers Improve Glycemia and Confer Cognitive Therapeutic Efficacy to Bound Insulin.

Diabetes poses a high risk for debilitating complications in neural tissues, regulating glucose uptake through insulin-dependent and predominantly insulin-independent pathways. Supramolecular nanostructures provide a flexible strategy for combinatorial regulation of glycemia. Here, we compare the effects of free insulin to insulin bound to positively charged nanofibers comprised of self-assembling amino acid compounds (AACs) with an antioxidant-modified side chain moiety (AAC2) in both in vitro and in vivo models of type 1 diabetes. Free AAC2, free human insulin (hINS) and AAC2-bound-human insulin (AAC2-hINS) were tested in streptozotocin (STZ)-induced mouse model of type 1 diabetes. AAC2-hINS acted as a complex and exhibited different properties compared to free AAC2 or hINS. Mice treated with the AAC2-hINS complex were devoid of hypoglycemic episodes, had improved levels of insulin in circulation and in the brain, and increased expression of neurotransmitter taurine transporter, Slc6a6. Consequently, treatment with AAC2-hINS markedly advanced both physical and cognitive performance in mice with STZ-induced and genetic type 1 diabetes compared to treatments with free AAC2 or hINS. This study demonstrates that the flexible nanofiber AAC2 can serve as a therapeutic platform for the combinatorial treatment of diabetes and its complications.

Alleviation of Metabolic Endotoxemia by Milk Fat Globule Membrane: Rationale, Design, and Methods of a Double-Blind, Randomized, Controlled, Crossover Dietary Intervention in Adults with Metabolic Syndrome.

BACKGROUND: Milk fat globule membrane (MFGM) is a phospholipid-rich component of dairy fat that might explain the benefits of full-fat dairy products on cardiometabolic risk. Preclinical studies support that MFGM decreases gut permeability, which could attenuate gut-derived endotoxin translocation and consequent inflammatory responses that impair cardiometabolic health. OBJECTIVES: To describe the rationale, study design, and planned outcomes that will evaluate the efficacy of MFGM-enriched milk compared with a comparator beverage on health-promoting gut barrier functions in persons with metabolic syndrome (MetS). METHODS: We plan a double-blind, randomized, crossover trial in which people with MetS will receive a rigorously controlled eucaloric diet for 2 wk that contains 3 daily servings of an MFGM-enriched bovine milk beverage or a comparator beverage that is formulated with nonfat dairy powder, coconut and palm oils, and soy phospholipids. Compliance will be monitored by assessing urinary para-aminobenzoic acid that is added to all test beverages. After the intervention, participants will ingest a high-fat/high-carbohydrate meal challenge to assess metabolic excursions at 30-min intervals for 3 h. Nondigestible sugar probes also will be ingested prior to collecting 24-h urine to assess region-specific gut permeability. Intervention efficacy will be determined based on circulating endotoxin (primary outcome) and glycemia (secondary outcome). Tertiary outcomes include: gut and systemic inflammatory responses, microbiota composition and SCFAs, gut permeability, and circulating insulin and incretins. EXPECTED RESULTS: MFGM is expected to decrease circulating endotoxin and glycemia without altering body mass. These improvements are anticipated to be accompanied by decreased gut permeability, decreased intestinal and circulating biomarkers of inflammation, increased circulating incretins, and beneficial antimicrobial and prebiotic effects in the gut microbiome. CONCLUSIONS: Demonstration of improvements in gut barrier functions that limit endotoxemia and glycemia could help to establish direct evidence that full-fat dairy lowers cardiometabolic risk, especially in people with MetS.The clinical trial associated with this article has been registered at clinicaltrials.gov (NCT03860584).

Amino acid-based compound activates atypical PKC and leptin receptor pathways to improve glycemia and anxiety like behavior in diabetic mice.

Differences in glucose uptake in peripheral and neural tissues account for the reduced efficacy of insulin in nervous tissues. Herein, we report the design of short peptides, referred as amino acid compounds (AAC) with and without a modified side chain moiety. At nanomolar concentrations, a candidate therapeutic molecule, AAC2, containing a 7-(diethylamino) coumarin-3-carboxamide side-chain improved glucose control in human peripheral adipocytes and the endothelial brain barrier cells by activation of insulin-insensitive glucose transporter 1 (GLUT1). AAC2 interacted specifically with the leptin receptor (LepR) and activated atypical protein kinase C zeta (PKCς) to increase glucose uptake. The effects induced by AAC2 were absent in leptin receptor-deficient predipocytes and in Leprdb mice. In contrast, AAC2 established glycemic control altering food intake in leptin-deficient Lepob mice. Therefore, AAC2 activated the LepR and acted in a cytokine-like manner distinct from leptin. In a monogenic Ins2Akita mouse model for the phenotypes associated with type 1 diabetes, AAC2 rescued systemic glucose uptake in these mice without an increase in insulin levels and adiposity, as seen in insulin-treated Ins2Akita mice. In contrast to insulin, AAC2 treatment increased brain mass and reduced anxiety-related behavior in Ins2Akita mice. Our data suggests that the unique mechanism of action for AAC2, activating LepR/PKCς/GLUT1 axis, offers an effective strategy to broaden glycemic control for the prevention of diabetic complications of the nervous system and, possibly, other insulin insensitive or resistant tissues.

Lactic acid bacteria isolated from dairy products as potential producers of lipolytic, proteolytic and antibacterial proteins.

Regular consumption of fermented dairy products helps maintain a healthy microbiota and prevent gut dysbiosis-linked diseases. The lactic acid bacteria (LAB) present in food enhance the digestibility of proteins, moderate the release of fatty acids, and support human health through inhabiting the gastrointestinal tract. These desirable properties of LAB are attributed, in part, to their metabolic processes involving enzymes such as lipases, proteases, and antibacterial proteins. The LAB strains presenting higher enzymatic activities may offer improved functionality for applications in foods. The first aim of this work was to isolate and identify LAB from diverse dairy products and select those with enhanced enzymatic activities. Secondly, this work aimed to investigate the subcellular organization and identity of these enzymes after semi-purification. Out of the total 137 LAB strains isolated and screened, 50.3% and 61.3% of the strains exhibited lipolytic and proteolytic activities, respectively. Seven strains displaying high enzymatic activities were selected and further characterized for the cellular organization of their lipases, proteases, and antibacterial proteins. The lipolytic and proteolytic activities were exhibited predominantly in the extracellular fraction; whereas, the antibacterial activities were found in various cellular fractions and were capable of inhibiting common undesirable microorganisms in foods. In total, two lipases, seven proteases, and three antibacterial proteins were identified by LC-MS/MS. Characterization of LAB strains with high enzymatic activity has potential biotechnological significance in fermentative processes and in human health as they may improve the physicochemical characteristics of foods and displace strains with weaker enzymatic activities in the human gut microbiota.

Symposium review: The relevance of bovine milk phospholipids in human nutrition-Evidence of the effect on infant gut and brain development.

This paper reflects the concepts reviewed during the presentation in the Joint MILK/Lactation Biology Symposium at the ADSA 2018 Annual Meeting. Our intention is to update the concepts and advances in the area of research regarding milk phospholipids or polar lipid fraction as part of a dairy ingredient used today in nutritional studies that focus on gut health as well as brain development of infants. Although processing advances have allowed the production of novel ingredients rich in milk fat globule membrane (MFGM) components, mostly monitored by phospholipid concentration and presence of membrane proteins, there is wide variability in their composition and structure. Furthermore, we aimed to include in the phospholipid fraction of milk nanovesicles designated as milk exosomes, which are secreted into milk by different secretion mechanisms than those of the fat globules but are also made up of a unique mixture of polar lipids. We consider imperative the study of polar lipid-derived structures from milk regarding composition and structure to gain insights into their biological effect in human health. Nevertheless, and tolerating the differences in composition and concentration of their components, studies supplementing the diet of infants with polar lipids (i.e., MFGM components) have shown significant advances in several areas of human health and well-being. Here we present a summary of the important components of MFGM and milk exosomes as well as an overview of the effects on gut health and brain and cognitive development when added to the diet of infants.

Production of bioactive conjugated linoleic acid by the multifunctional enolase from Lactobacillus plantarum.

Lactobacillus plantarum α-enolase, a multifunctional-anchorless-surface protein belonging to the conserved family of enolases with a central role in glycolytic metabolism, was characterized to have a side role in the intricate metabolism of biohydrogenation of linoleic acid, catalyzing the formation of bioactive 9-cis-11-trans-CLA through dehydration and isomerization of 10-hydroxy-12-cis-octadecenoic acid. The identity of the enolase was confirmed through mass spectrometric analysis that showed the characteristic 442 amino acid sequence with a molecular mass of 48.03kDa. The enolase was not capable of using linoleic acid directly as a substrate but instead uses its hydroxyl derivative 10-hydroxi-12-cis-octadecenoic acid to finally form bioactive conjugated linoleic acid. Biochemical optimization studies were carried out to elucidate the conditions for maximum production of 9-cis-11-trans-CLA and maximum stability of α-enolase when catalyzing this reaction. Furthermore, through structural analysis of the protein, we propose the binding sites of substrate and product molecules that were characterized as two hydrophobic superficial pockets located at opposite ends of the enolase connected through a channel where the catalysis of dehydration and isomerization might occur. These results prove that multifunctional α-enolase also plays a role in cell detoxification from polyunsaturated fatty acids such as linoleic acid, along with the linoleate isomerase complex.

Functional characterization of a fatty acid double-bond hydratase from Lactobacillus plantarum and its interaction with biosynthetic membranes.

Hydrogenation of linoleic acid and other polyunsaturated fatty acids is a detoxification mechanism that is present in the Lactobacillus genus of lactic bacteria. The first stage in this multi-step process is hydration of the substrate with formation of 10-hydroxy-9-cis-octadecenoic acid due to fatty-acid hydratase activity that has been detected only in the membrane-associated cell fraction; however, its interaction with the cell membrane is unknown. To provide information in this respect we characterized the homotrimeric 64.7 kDa-native protein from Lactobacillus plantarum; afterwards, it was reconstituted in proteoliposomes and analyzed by confocal fluorescence microscopy. The results showed that hydratase is an extrinsic-membrane protein and hence, the enzymatic reaction occurs at the periphery of the cell. This location may be advantageous in the detoxifying process since the toxic linoleic acid molecule can be bound to hydratase and converted to non-toxic 10-hydroxy-9-cis-octadecenoic acid before it reaches cell membrane. Additionally, we propose that the interaction with membrane periphery occurs through electrostatic contacts. Finally, the structural model of L. plantarum hydratase was constructed based on the amino acid sequence and hence, the putative binding sites with linoleic acid were identified: site 1, located in an external hydrophobic pocket at the C-terminus of the protein and site 2, located at the core and in contact with a FAD molecule. Interestingly, it was found that the linoleic acid molecule arranges around a methionine residue in both sites (Met154 and Met81, respectively) that acts as a rigid pole, thus playing a key role in binding unsaturated fatty acids.