Evaluation of Extraction Techniques for Recovery of Microalgal Lipids under Different Growth Conditions.

Microalgal lipids contain a wide array of liposoluble bioactive compounds, but lipid extraction remains a critical limitation for their commercial use. An accelerated solvent extraction (ASE) was used to extract lipids from Chlamydomonas reinhardtii, Arthrospira platensis (Spirulina), and Chlorella vulgaris grown under either standard or nitrogen depletion conditions. Under standard growth conditions, ASE using methanol:chloroform (2:1), methyl tert-butyl ether (MTBE):methanol:water, and ethanol at 100 °C resulted in the highest recovery of total lipids (352 ± 30, 410 ± 32, and 127 ± 15 mg/g biomass from C. reinhardtii, C. vulgaris, and A. platensis, respectively). Similarly, the highest total lipid and triacylglycerols (TAGs) recovery from biomass cultivated under nitrogen depletion conditions was found at 100 °C using methanol:chloroform, for C. reinhardtii (total, 550 ± 21; TAG, 205 ± 2 mg/g biomass) and for C. vulgaris (total, 612 ± 29 mg/g; TAG, 253 ± 7 mg/g biomass). ASE with MTBE:methanol:water at 100 °C yielded similar TAG recovery for C. reinhardtii (159 ± 6 mg/g) and C. vulgaris (200 ± 4 mg/g). Thus, MTBE:methanol:water is suggested as an alternative substitute to replace hazardous solvent mixtures for TAGs extraction with a much lower environmental impact. The extracted microalgal TAGs were rich in palmitic (C16:0), stearic (C18:0), oleic (C18:1,9), linoleic (C18:2n6), and α-linolenic (C18:3n3) acids. Under nitrogen depletion conditions, increased palmitic acid (C16:0) recovery up to 2-fold was recorded from the biomasses of C. reinhardtii and C. vulgaris. This study demonstrates a clear linkage between the extraction conditions applied and total lipid and TAG recovery.

A Diet Enriched with Lacticaseibacillus rhamnosus HN001 and Milk Fat Globule Membrane Alters the Gut Microbiota and Decreases Amygdala GABA a Receptor Expression in Stress-Sensitive Rats.

Brain signalling pathways involved in subclinical anxiety and depressed mood can be modulated via the gut brain axis (GBA), providing the potential for diet and dietary components to affect mood. We investigated behavioural, physiological and gut microbiome responses to the Lacticaseibacillus rhamnosus strain HN001 (LactoB HN001™), which has been shown to reduce postpartum anxiety and depression, and a milk fat globule membrane-enriched product, Lipid 70 (SurestartTM MFGM Lipid 70), which has been implicated in memory in stress-susceptible Wistar Kyoto rats. We examined behaviour in the open field, elevated plus maze and novel object recognition tests in conjunction with the expression of host genes in neuro-signalling pathways, and we also assessed brain lipidomics. Treatment-induced alterations in the caecal microbiome and short-chain fatty acid (SCFA) profiles were also assessed. Neither ingredient induced behavioural changes or altered the brain lipidome (separately or when combined). However, with regard to brain gene expression, the L. rhamnosus HN001 + Lipid 70 combination produced a synergistic effect, reducing GABAA subunit expression in the amygdala (Gabre, Gat3, Gabrg1) and hippocampus (Gabrd). Treatment with L. rhamnosus HN001 alone altered expression of the metabotropic glutamate receptor (Grm4) in the amygdala but produced only minor changes in gut microbiota composition. In contrast, Lipid 70 alone did not alter brain gene expression but produced a significant shift in the gut microbiota profile. Under the conditions used, there was no observed effect on rat behaviour for the ingredient combination. However, the enhancement of brain gene expression by L. rhamnosus HN001 + Lipid 70 implicates synergistic actions on region-specific neural pathways associated with fear, anxiety, depression and memory. A significant shift in the gut microbiota profile also occurred that was mainly attributable to Lipid 70.

The fortification of encapsulated soy isoflavones and texture modification of soy milk by α-lactalbumin nanotubes.

Nanocarriers can improve the dispersibility of hydrophobic bioactive compounds and potentially improve the texture of liquid food formulations. Here, nanotubes (NTs) with a high aspect ratio formed by self-assembly of peptides partially hydrolyzed from α-lactalbumin (α-lac) were used to deliver soy isoflavones (IFs) and modify soy milk texture. IFs encapsulated by nanotube (NT/IFs) via hydrophobic interactions, which had improved dispersibility, with a maximum loading efficiency of 4%. The rheological characterization showed that the nanotubes enhanced the viscoelastic property and long term-stability of soy milk. About 80% of the NT/IFs in soy milk survived simulated in in vitro gastric digestion promoting the release of IFs in the intestinal phase. Overall, this work demonstrated that α-lac nanotubes may be a multi-functional carrier system for hydrophobic compounds providing beneficial changes to functional food texture.

Variation in milk fat globule size and composition: A source of bioactives for human health.

Milk fat globules (MFGs) are secreted from the mammalian gland and are composed of a triacylglycerol core surrounded by a triple membrane structure, the milk fat globule membrane (MFGM). The MFGM contains complex lipids and proteins reported to have nutritional, immunological, neurological and digestive functions. Human and ruminant milk are shown to share a similar MFG structure but with different size, profile and abundance of protein and polar lipids. This review summarizes the reported data on human, bovine, caprine and ovine MFG composition and concentration of bioactive components in different MFG-size fractions. A comprehensive understanding of compositional variations between milk from different species and MFG size fractions may help promote various milk sources as targeted supplements to improve human development and health. MFG size and MFGM composition are species-specific and affected by lactation, diet and breed (or maternal origin). Purification and enrichment methods for some bioactive proteins and lipids present in the MFGM have yet to be established or are not scaled sufficiently to be used to supplement human diets. To overcome this problem, MFG size selection through fractionation or herd selection may provide a convenient way to pre-enrich the MFG fraction with specific protein and lipid components to fulfill human dietary and health requirements.

Changes in Human Milk Fat Globule Composition Throughout Lactation: A Review.

There has been a growing interest in understanding how the relative levels of human milk fat globule (MFG) components change over the course of lactation, how they differ between populations, and implications of these changes for the health of the infant. In this article, we describe studies published over the last 30 years which have investigated components of the MFG in term milk, focusing on changes over the course of lactation and highlighting infant and maternal factors that may influence these changes. We then consider how the potential health benefits of some of the milk fat globule membrane (MFGM) components and derived ingredients relate to compositional and functional aspects and how these change throughout lactation. The results show that the concentrations of phospholipids, gangliosides, cholesterol, fatty acids and proteins vary throughout lactation, and such changes are likely to reflect the changing requirements of the growing infant. There is a lack of consistent trends for changes in phospholipids and gangliosides across lactation which may reflect different methodological approaches. Other factors such as maternal diet and geographical location have been shown to influence human MFGM composition. The majority of research on the health benefits of MFGM have been conducted using MFGM ingredients derived from bovine milk, and using animal models which have clearly demonstrated the role of the MFGM in supporting cognitive and immune health of infants at different stages of growth and development.

Changes in HMO Concentrations throughout Lactation: Influencing Factors, Health Effects and Opportunities.

Human milk oligosaccharides (HMOs) are important functional biomolecules in human breast milk. Understanding the factors influencing differences in HMO composition and changes in their concentration over lactation can help to design feeding strategies that are well-adapted to infant's needs. This review summarises the total and individual concentration of HMOs from data published from 1999 to 2019. Studies show that the HMO concentrations are highest in colostrum (average 9-22 g/L), followed by slightly lower concentrations in transitional milk (average 8-19 g/L), with a gradual decline in mature milk as lactation progresses, from 6-15 g/L in breast milk collected within one month of birth, to 4-6 g/L after 6 months. Significant differences in HMO composition have been described between countries. Different HMOs were shown to be predominant over the course of lactation, e.g., 3-fucosyllactose increased over lactation, whereas 2'-fucosyllactose decreased. Recent clinical studies on infant formula supplemented with 2'-fucosyllactose in combination with other oligosaccharides showed its limited beneficial effect on infant health.

In vitro Fermentation of Digested Milk Fat Globule Membrane From Ruminant Milk Modulates Piglet Ileal and Caecal Microbiota.

Lipids in milk are secreted as a triacylglycerol core surrounded by a trilayer membrane, the milk fat globule membrane (MFGM). This membrane, known to have important roles in infant brain and intestinal development, is composed of proteins, glycoproteins, and complex lipids. We hypothesized that some of the beneficial properties of MFGM are due to its effects on the gastrointestinal microbiota. This study aimed to determine the effect of a commercial phospholipid concentrate (PC) and enriched bovine, caprine, and ovine MFGM fractions on ileal and hindgut microbiota in vitro. Digestion of PC and MFGMs was conducted using an in vitro model based on infant gastric and small intestine conditions. The recovered material was then in vitro fermented with ileal and caecal inocula prepared from five piglets fed a commercial formula for 20 days before ileal and caecal digesta were collected. After each fermentation, samples were collected to determine organic acid production and microbiota composition using 16S rRNA sequencing. All substrates, except PC (5%), were primarily fermented by the ileal microbiota (8-14%) (P < 0.05). PC and caprine MFGM reduced ileal microbiota alpha diversity compared to ileal inoculum. Caprine MFGM increased and PC reduced the ileal ratio of firmicutes:proteobacteria (P < 0.05), respectively, compared to the ileal inoculum. Bovine and ovine MFGMs increased ileal production of acetic, butyric, and caproic acids compared to other substrates and reduced the proportions of ileal proteobacteria (P < 0.0001). There was a limited fermentation of bovine (3%), caprine (2%), and ovine (2%) MFGMs by the caecal microbiota compared to PC (14%). In general, PC and all MFGMs had a reduced effect on caecal microbiota at a phylum level although MFG source-specific effects were observed at the genus level. These indicate that the main effects of the MFGM in the intestinal microbial population appears to occur in the ileum.

Understanding the Effects of Lactose Hydrolysis Modeling on the Main Oligosaccharides in Goat Milk Whey Permeate.

Enzymatic hydrolysis of lactose is a crucial step to improve the efficiency and selectivity of membrane-based separations toward the recovery of milk oligosaccharides free from simple sugars. Response surface methodology was used to investigate the effects temperature (25.9 to 54.1 °C) and amount of enzyme (0.17 to 0.32% w/w) at 1, 2, and 4 h of reaction on the efficiency of lactose hydrolysis by Aspergillus oryzae ß-galactosidase, preservation of major goat whey oligosaccharides, and on the de-novo formation of oligosaccharides. Lactose hydrolysis above 99% was achieved at 1, 2, and 4 h, not being significantly affected by temperature and amount of enzyme within the tested conditions. Formation of 4 Hexose (Hex) and 4 Hex 1 Hex and an increased de-novo formation of 2 Hex 1 N-Acetyl-Neuraminic Acid (NeuAc) and 2 Hex 1 N-Glycolylneuraminic acid (NeuGc) was observed in all treatments. Overall, processing conditions using temperatures ≤40 °C and enzyme concentration ≤0.25% resulted in higher preservation/formation of goat whey oligosaccharides.

Effects of Prenatal Consumption of Caprine Milk Oligosaccharides on Mice Mono-associated with Bifidobacterium Bifidum (AGR2166).

BACKGROUND: Prenatal consumption of oligosaccharides are associated with changes in the maternal gastrointestinal tract (GIT) microbiota with health consequences for the offspring. It has previously been demonstrated that caprine milk oligosaccharides (CMO) stimulate the growth and fermentation rate of Bifidobacterium bifidum AGR2166. OBJECTIVE: The objective of this study was to examine the effects of B. bifidum AGR2166 and prenatal consumption of CMO, alone or in combination, on the dam's large intestine, foetal development and ability of B. bifidum to translocate from the gastrointestinal lumen to organs and foetal membranes. METHOD: Germ-free BALB/c mice, inoculated with B. bifidum AGR2166 or anaerobic phosphate buffer, were fed either diet supplemented with CMO or with galacto-oligosaccharide. Pregnant mice were euthanised 1 to 3 days before the expected delivery date and samples collected for analysis. RESULTS: Dietary CMO, regardless of bifidobacterial inoculation was shown to increase GIT weight and to reduce foetal weight compared to galacto-oligosaccharide-fed dams. B. bifidum AGR2166 DNA was detected in the mesenteric lymph nodes, liver, plasma and placenta of the dam by amplification of the bifidobacterial 16S rRNA gene. CONCLUSION: B. bifidum AGR2166 DNA was detected in maternal organs, however there is no indication that live bifidobacteria was able to translocate during pregnancy. Further studies using conventionally-raised mouse models will develop a deeper understanding of the interactions between dietary CMOF, the host, and bacteria.

Prenatal caprine milk oligosaccharide consumption affects the development of mice offspring.

SCOPE: The composition of the gastrointestinal (GIT) microbiota, particularly in early life, influences the development of metabolic diseases later in life. The maternal microbiota is the main source of bacteria colonising the infant GIT and can be modified by dietary prebiotics. Our objective was to determine the effects of prenatal consumption of prebiotic caprine milk oligosaccharides (CMO) on the large intestine of female mice, milk composition, and offspring's development. METHODS AND RESULTS: C57BL/6 mice were fed either a control diet, CMO diet, or galacto-oligosaccharide diet from mating to weaning. From weaning, some pups nursed by CMO, GOS, and control-dams were fed the control diet for 30 days. CMO or GOS-fed dams had increased colon length and milk protein concentration compared to control-fed dams. At weaning, pups from CMO-fed dams had increased body weight and colon length and increased proportions of colonic Bifidobacterium spp compared to the pups from control-fed dams. Thirty days after weaning, pups from CMO-fed dams had increased visceral fat weight compared to pups from control-fed dams. CONCLUSION: Consumption of CMO by the dams during gestation and lactation improved the development of the pups, and the relative abundance of bifidobacteria and butyric acid in the colon, at weaning.

In Vitro Fermentation of caprine milk oligosaccharides by bifidobacteria isolated from breast-fed infants.

This study was conducted to investigate the catabolism and fermentation of caprine milk oligosaccharides (CMO) by selected bifidobacteria isolated from 4 breast-fed infants. Seventeen bifidobacterial isolates consisting of 3 different species (Bifidobacterium breve, Bifidobacterium longum subsp. longum and Bifidobacterium bifidum) were investigated. A CMO-enriched fraction (CMOF) (50% oligosaccharides, 10% galacto-oligosaccharides (GOS), 20% lactose, 10% glucose and 10% galactose) from caprine cheese whey was added to a growth medium as a sole source of fermentable carbohydrate. The inclusion of the CMOF was associated with increased bifidobacterial growth for all strains compared to glucose, lactose, GOS, inulin, oligofructose, 3'-sialyl-lactose and 6'-sialyl-lactose. Only one B. bifidum strain (AGR2166) was able to utilize the sialyl-CMO, 3'-sialyl-lactose and 6'-sialyl-lactose, as carbohydrate sources. The inclusion of CMOF increased the production of acetic and lactic acid (P < 0.001) after 36 h of anaerobic fermentation at 37 °C, when compared to other fermentable substrates. Two B. bifidum strains (AGR2166 and AGR2168) utilised CMO, contained in the CMOF, to a greater extent than B. breve or B. longum subsp longum isolates, and this increased CMO utilization was associated with enhanced sialidase activity. CMOF stimulated bifidobacterial growth when compared to other tested fermentable carbohydrates and also increased the consumption of mono- and disaccharides, such as galactose and lactose present in the CMOF. These findings indicate that the dietary consumption of CMO may stimulate the growth and metabolism of intestinal Bifidobacteria spp. including B. bifidum typically found in the large intestine of breast-fed infants.

Can nutritional modulation of maternal intestinal microbiota influence the development of the infant gastrointestinal tract?

The gastrointestinal microbiota plays an important role in maintaining host health by preventing the colonization of pathogens, fermenting dietary compounds, and maintaining normal mucosal immunity. Particularly in early life, the composition of the microbiota profoundly influences the development and maturation of the gastrointestinal tract (GIT) mucosa, which may affect health in later life. Therefore, strategies to manipulate the microbiota during infancy may prevent the development of some diseases later in adult life. Earlier research suggested that term fetuses are sterile and that the initial bacterial colonization of the newborn GIT occurs only after the baby transits through the birth canal. However, recent studies have demonstrated that the colonization and/or contact of the fetus with the maternal GIT microbiota may start in utero. After vaginal birth, the colonization of the neonate GIT continues through contact with maternal feces and vaginal bacteria, leading to a relatively simple microbial community that is influenced by feeding type (breast vs. formula feeding). Maternal GIT microbiota, vaginal microbiota, and breast milk composition are influenced by maternal diet. Alterations of the maternal GIT microbiota composition via supplementation with probiotics and prebiotics have been shown; however, transfer of these benefits to the offspring remains to be demonstrated. This review focuses on the influence of maternal GIT microbiota during the pre- and postpartum periods on the colonization of the infant GIT. In particular, it examines the manipulation of the maternal GIT microbiota composition through the use of probiotics and/or prebiotics and subsequent consequences for the health of the offspring.

The Rv1712 Locus from Mycobacterium tuberculosis H37Rv codes for a functional CMP kinase that preferentially phosphorylates dCMP.

The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed.

New sensitive fluorophores for selective DNA detection.

4,7-Disubstituted benzothiadiazoles containing 1-arylethynyl and 4-methoxyphenyl groups are selective photoluminescent "light up" probes to duplex DNA with unprecedented sensibility in both spectrophotometric and spectrofluorimetric measurements.