Synthesis of N-Acetyllactosamine and N-Acetyllactosamine-Based Bioactives.

N-Acetyllactosamine (LacNAc) or more specifically ß-d-galactopyranosyl-1,4-N-acetyl-d-glucosamine is a unique acyl-amino sugar and a key structural unit in human milk oligosaccharides, an antigen component of many glycoproteins, and an antiviral active component for the development of effective drugs against viruses. LacNAc is useful itself and as a basic building block for producing various bioactive oligosaccharides, notably because this synthesis may be used to add value to dairy lactose. Despite a significant amount of information in the literature on the benefits, structures, and types of different LacNAc-derived oligosaccharides, knowledge about their effective synthesis for large-scale production is still in its infancy. This work provides a comprehensive analysis of existing production strategies for LacNAc and important LacNAc-based structures, including sialylated LacNAc as well as poly- and oligo-LacNAc. We conclude that direct extraction from milk is too complex, while chemical synthesis is also impractical at an industrial scale. Microbial routes have application when multiple step reactions are needed, but the major route to large-scale biochemical production will likely lie with enzymatic routes, particularly those using ß-galactosidases (for LacNAc synthesis), sialidases (for sialylated LacNAc synthesis), and ß-N-acetylhexosaminidases (for oligo-LacNAc synthesis). Glycosyltransferases, especially for the biosynthesis of extended complex LacNAc structures, could also play a major role in the future. In these cases, immobilization of the enzyme can increase stability and reduce cost. Processing parameters, such as substrate concentration and purity, acceptor/donor ratio, water activity, and temperature, can affect product selectivity and yield. More work is needed to optimize these reaction parameters and in the development of robust, thermally stable enzymes to facilitate commercial production of these important bioactive substances.

Single and binary ion sorption equilibria of monovalent and divalent ions in commercial ion exchange membranes.

The co-ion and counter-ion sorption of monovalent (Na+, K+, Cl- and NO3-) and divalent ions (Ca2+ and SO42-) in commercial Neosepta ion exchange membranes were systemically studied in both single and binary salt systems. The new generation of Neosepta cation exchange membrane (CSE) showed a significant difference in water uptake and co-ion sorption compared to the earlier generation (CMX). Use of the Manning model confirmed that there were significant differences between these membranes, with the estimated value of the Manning parameter changing from 1.0 ± 0.1 for CMX to 2.8 ± 0.5 for CSE. There were fewer differences between the two Neosepta anion exchange membranes, AMX and ASE. In single salt solutions, potassium sorbed most strongly into the cation exchange membranes, but in binary salt mixtures, calcium dominated due to Donnan exclusion at low concentrations. While these trends were expected, the sorption behaviour in the anion exchange membranes was more complex. The water uptake of both AMX and ASE was shown to be the greatest in Na2SO4 solutions. This strong water uptake was reflected in strong sorption of sulphate ions in a single salt solution. Conversely, in a binary salt mixture with NaCl, sulphate sorption fell significantly at higher concentrations. This was possibly caused by ion pairing within the solution, as well as the strongly hydrophobic nature of styrene in the charged polymer. Water uptake was lowest in NaNO3 solutions, even though sorption of the nitrate ion was comparable to that of chloride in these single salt solutions. In the binary mixture, nitrate was absorbed more strongly than chloride. These results could be due to the low surface charge density of this ion allowing it to bond more strongly with the hydrophobic polymeric backbone at the exclusion of water and other ions.

A review of salty waste stream management in the Australian dairy industry.

Saline wastewater is a by-product of cheese manufacturing and whey processing that can have serious environmental and economic consequences. Salty streams originating from dairy processing operations include chromatography wastes, clean-in-place wastewater, acid whey, salty whey and waste generated from whey demineralization processes such as nanofiltration, electrodialysis and ion exchange. With the participation of the major dairy companies in Australia, an industry wide survey was conducted to acquire a comprehensive understanding of the management strategies for these salty waste streams. High salinity waste streams are commonly directed to evaporation ponds. However, environmental impacts from land degradation, odour and dust have prevented the construction of further evaporation ponds in some areas of Australia. The survey results also show that disposal to municipal trade waste is not always effective, as the current levels of some salinity-related parameters are significantly higher than the levels allowed by the local water/environmental authorities. For high salinity streams, salt removal can lead to more substantial savings in trade waste charges compared to wastewater volume reduction. Thus, salt removal and recovery from salty waste streams has become a major focus of the sustainability agenda of the Australian dairy industry.

The role of neuropeptide W in energy homeostasis.

Neuropeptide W is the endogenous ligand for G-protein-coupled receptors GPR7 and GPR8. In this review, we summarize findings on the distribution of neuropeptide W and its receptors in the central nervous system and the periphery, and discuss the role of NPW in food intake and energy homeostasis.

Upper Gastrointestinal Function in Morbidly Obese Adolescents Before and 6 Months After Gastric Banding.

BACKGROUND: The effects of laparoscopic adjustable gastric band (LAGB) placement on upper gastrointestinal tract function in obese adolescents are unknown. Therefore, our aim was to determine the short-term effects of LAGB on esophageal motility, gastroesophageal reflux, gastric emptying, appetite-regulatory hormones, and perceptions of post-prandial hunger and fullness. METHODS: This study was part of a prospective cohort study (March 2009-December 2015) in one tertiary referral hospital. The study included obese adolescents (14-18 years) with a body mass index (BMI) > 40 (or ≥ 35 with comorbidities). Gastric emptying was assessed by 13C-octanoic acid breath test, pharyngeal, and esophageal motor function by high-resolution manometry with impedance (HRIM), and appetite and other perceptions using 100-mm visual analogue scales. Dysphagia symptoms were scored using a Dakkak questionnaire. Data were compared pre- and post-LAGB placement and at a 6-month follow-up. RESULTS: Based upon analysis of 15 adolescents, at the 6-month follow-up, LAGB placement: (i) led to a significant reduction in weight and BMI; (ii) increased fullness and decreased hunger post-meal; (iii) increased symptoms of dysphagia after solid food; and, despite these effects, (iv) caused little or no changes to appetite hormones, while (v) effects on gastric emptying, esophageal motility, esophageal bolus transport, and esophageal emptying were not significant. CONCLUSION: In adolescents, LAGB improved BMI and altered the sensitivity to nutrients without significant effects on upper gastrointestinal tract physiology at the 6-month follow-up.

Solubility of Calcium Phosphate in Concentrated Dairy Effluent Brines.

The solubility of calcium phosphate in concentrated dairy brine streams is important in understanding mineral scaling on equipment, such as membrane modules, evaporators, and heat exchangers, and in brine pond operation. In this study, the solubility of calcium phosphate has been assessed in the presence of up to 300 g/L sodium chloride as well as lactose, organic acids, and anions at 10, 30, and 50 °C. As a neutral molecule, lactose has a marginal but still detectable effect upon calcium solubility. However, additions of sodium chloride up to 100 g/L result in a much greater increase in calcium solubility. Beyond this point, the concentrations of ions in the solution decrease significantly. These changes in calcium solubility can readily be explained through changes in the activity coefficients. There is little difference in calcium phosphate speciation between 10 and 30 °C. However, at 50 °C, the ratio of calcium to phosphate in the solution is lower than at the other temperatures and varies less with ionic strength. While the addition of sodium lactate has less effect upon calcium solubility than sodium citrate, it still has a greater effect than sodium chloride at an equivalent ionic strength. Conversely, when these organic anions are present in the solution in the acid form, the effect of pH dominates and results in much higher solubility and a calcium/phosphate ratio close to one, indicative of dicalcium phosphate dihydrate as the dominant solid phase.

Plasticity of gastrointestinal vagal afferent satiety signals.

The vagal link between the gastrointestinal tract and the central nervous system (CNS) has numerous vital functions for maintaining homeostasis. The regulation of energy balance is one which is attracting more and more attention due to the potential for exploiting peripheral hormonal targets as treatments for conditions such as obesity. While physiologically, this system is well tuned and demonstrated to be effective in the regulation of both local function and promoting/terminating food intake the neural connection represents a susceptible pathway for disruption in various disease states. Numerous studies have revealed that obesity in particularly is associated with an array of modifications in vagal afferent function from changes in expression of signaling molecules to altered activation mechanics. In general, these changes in vagal afferent function in obesity further promote food intake instead of the more desirable reduction in food intake. It is essential to gain a comprehensive understanding of the mechanisms responsible for these detrimental effects before we can establish more effective pharmacotherapies or lifestyle strategies for the treatment of obesity and the maintenance of weight loss.

Sex-specific alterations in glucose homeostasis and metabolic parameters during ageing of caspase-2-deficient mice.

Gender-specific differences are commonly found in metabolic pathways and in response to nutritional manipulation. Previously, we identified a role for caspase-2 in age-related glucose homeostasis and lipid metabolism using male caspase-2-deficient (Casp2 (-/-) ) mice. Here we show that the resistance to age-induced glucose tolerance does not occur in female Casp2 (-/-) mice and it appears to be independent of insulin sensitivity in males. Using fasting (18 h) as a means to further investigate the role of caspase-2 in energy and lipid metabolism, we identified sex-specific differences in the fasting response and lipid mobilization. In aged (18-22 months) male Casp2 (-/-) mice, a significant decrease in fasting liver mass, but not total body weight, was observed while in females, total body weight, but not liver mass, was reduced when compared with wild-type (WT) animals. Fasting-induced lipolysis of adipose tissue was enhanced in male Casp2 (-/-) mice as indicated by a significant reduction in white adipocyte cell size, and increased serum-free fatty acids. In females, white adipocyte cell size was significantly smaller in both fed and fasted Casp2 (-/-) mice. No difference in fasting-induced hepatosteatosis was observed in the absence of caspase-2. Further analysis of white adipose tissue (WAT) indicated that female Casp2 (-/-) mice may have enhanced fatty acid recycling and metabolism with expression of genes involved in glyceroneogenesis and fatty acid oxidation increased. Loss of Casp2 also increased fasting-induced autophagy in both male and female liver and in female skeletal muscle. Our observations suggest that caspase-2 can regulate glucose homeostasis and lipid metabolism in a tissue and sex-specific manner.

Crystallisation of minerals from concentrated saline dairy effluent.

An understanding of crystallisation within saline effluents is important for the design of both brine crystallisers and brine disposal ponds. In this work, crystallisation of a saline effluent concentrate from the Australian dairy industry has been examined at 22 wt% and 30 wt% total solids and at temperatures between 10 and 70 °C. The precipitation occurs more rapidly at higher temperatures. This trend is dictated by precipitation of calcium phosphate salts, albeit the major constituents of the mixture are NaCl and lactose. The crystallisation induction time can be shortened by introducing cavitation induced by ultrasound. In particular, the use of two short acoustic pulses between 3.7 J/g and 16 J/g at 20 kHz spaced ten minutes apart has maximum impact upon both induction time and crystal size. It is believed that the first ultrasound pulse either generates new nuclei or enhances the mass transfer of solute toward the surface of sub-micron growing crystals. Conversely, the second pulse disrupts the growing crystals and forms secondary nuclei. The ultrasound cannot shift the solution equilibrium and so is not able to improve the low crystal yield. To increase this total yield, further evaporation is necessary. The work provides direction to personnel in the dairy industry of the feasibility of brine crystallisation with respect to energy demand and solid recovery.

Inflammasome signaling affects anxiety- and depressive-like behavior and gut microbiome composition.

The inflammasome is hypothesized to be a key mediator of the response to physiological and psychological stressors, and its dysregulation may be implicated in major depressive disorder. Inflammasome activation causes the maturation of caspase-1 and activation of interleukin (IL)-1ß and IL-18, two proinflammatory cytokines involved in neuroimmunomodulation, neuroinflammation and neurodegeneration. In this study, C57BL/6 mice with genetic deficiency or pharmacological inhibition of caspase-1 were screened for anxiety- and depressive-like behaviors, and locomotion at baseline and after chronic stress. We found that genetic deficiency of caspase-1 decreased depressive- and anxiety-like behaviors, and conversely increased locomotor activity and skills. Caspase-1 deficiency also prevented the exacerbation of depressive-like behaviors following chronic stress. Furthermore, pharmacological caspase-1 antagonism with minocycline ameliorated stress-induced depressive-like behavior in wild-type mice. Interestingly, chronic stress or pharmacological inhibition of caspase-1 per se altered the fecal microbiome in a very similar manner. When stressed mice were treated with minocycline, the observed gut microbiota changes included increase in relative abundance of Akkermansia spp. and Blautia spp., which are compatible with beneficial effects of attenuated inflammation and rebalance of gut microbiota, respectively, and the increment in Lachnospiracea abundance was consistent with microbiota changes of caspase-1 deficiency. Our results suggest that the protective effect of caspase-1 inhibition involves the modulation of the relationship between stress and gut microbiota composition, and establishes the basis for a gut microbiota-inflammasome-brain axis, whereby the gut microbiota via inflammasome signaling modulate pathways that will alter brain function, and affect depressive- and anxiety-like behaviors. Our data also suggest that further elucidation of the gut microbiota-inflammasome-brain axis may offer novel therapeutic targets for psychiatric disorders.

Altered gastric vagal mechanosensitivity in diet-induced obesity persists on return to normal chow and is accompanied by increased food intake.

BACKGROUND AND AIMS: Gastric vagal afferents convey satiety signals in response to mechanical stimuli. The sensitivity of these afferents is decreased in diet-induced obesity. Leptin, secreted from gastric epithelial cells, potentiates the response of vagal afferents to mechanical stimuli in lean mice, but has an inhibitory effect in high-fat diet (HFD)-induced obese mice. We sought to determine whether changes in vagal afferent function and response to leptin in obesity were reversible by returning obese mice consuming a HFD to standard laboratory chow diet (SLD). METHODS: Eight-week-old female C57BL/6 mice were either fed a SLD (N=20) or HFD (N=20) for 24 weeks. A third group was fed a HFD for 12 weeks and then a SLD for a further 12 weeks (RFD, N=18). An in vitro gastro-oesophageal vagal afferent preparation was used to determine the mechanosensitivity of gastric vagal afferents and the modulatory effect of leptin (0.1-10 nM) was examined. Retrograde tracing and quantitative RT-PCR were used to determine the expression of leptin receptor (LepR) messenger RNA (mRNA) in whole nodose and specific cell bodies traced from the stomach. RESULTS: After 24 weeks, both the HFD and RFD mice had increased body weight, gonadal fat mass, plasma leptin, plasma insulin and daily energy consumption compared with the SLD mice. The HFD and RFD mice had reduced tension receptor mechanosensitivity and leptin further inhibited responses to tension in HFD, RFD but not SLD mice. Mucosal receptors from both the SLD and RFD mice were potentiated by leptin, an effect not seen in HFD mice. LepR expression was unchanged in the whole nodose, but was reduced in the mucosal afferents of the HFD and RFD mice. CONCLUSION: Disruption of gastric vagal afferent function by HFD-induced obesity is only partially reversible by dietary change, which provides a potential mechanism preventing maintenance of weight loss.

Modulation of murine gastric vagal afferent mechanosensitivity by neuropeptide W.

AIM: Neuropeptide W (NPW) is an endogenous ligand for the receptors GPR7 and GPR8 and is involved in central regulation of energy homeostasis. NPW in the periphery is found in gastric gastrin (G) cells. In the stomach, energy intake is influenced by vagal afferent signals, so we aimed to determine the effect of NPW on mechanosensitive gastric vagal afferents under different feeding conditions. METHODS: Female C57BL/6 mice (N > 10 per group) were fed a standard laboratory diet (SLD), high-fat diet (HFD) or were food restricted. The relationship between NPW immunopositive cells and gastric vagal afferent endings was determined by anterograde tracing and NPW immunohistochemistry. An in vitro gastro-oesophageal preparation was used to determine the functional effects of NPW on gastric vagal afferents. Expression of NPW in the gastric mucosa and GPR7 in whole nodose ganglia was determined by quantitative RT-PCR (QRT-PCR). The expression of GPR7 in gastric vagal afferent neurones was determined by retrograde tracing and QRT-PCR. RESULTS: Neuropeptide W immunoreactive cells were found in close proximity to traced vagal afferents. NPW selectively inhibited responses of gastric vagal tension receptors to stretch in SLD but not HFD or fasted mice. In the nodose ganglia, GPR7 mRNA was specifically expressed in gastric vagal afferent neurones. In fasted mice gastric mucosal NPW and nodose GPR7, mRNA was reduced compared with SLD. A HFD had no effect on gastric NPW mRNA, but down-regulated nodose GPR7 expression. CONCLUSION: Neuropeptide W modulates gastric vagal afferent activity, but the effect is dynamic and related to feeding status.

The effect of ultrasound on casein micelle integrity.

Samples of fresh skim milk, reconstituted micellar casein, and casein powder were sonicated at 20 kHz to investigate the effect of ultrasonication. For fresh skim milk, the average size of the remaining fat globules was reduced by approximately 10 nm after 60 min of sonication; however, the size of the casein micelles was determined to be unchanged. A small increase in soluble whey protein and a corresponding decrease in viscosity also occurred within the first few minutes of sonication, which could be attributed to the breakup of casein-whey protein aggregates. No measurable changes in free casein content could be detected in ultracentrifuged skim milk samples sonicated for up to 60 min. A small, temporary decrease in pH resulted from sonication; however, no measurable change in soluble calcium concentration was observed. Therefore, casein micelles in fresh skim milk were stable during the exposure to ultrasonication. Similar results were obtained for reconstituted micellar casein, whereas larger viscosity changes were observed as whey protein content was increased. Controlled application of ultrasound can be usefully applied to reverse process-induced protein aggregation without affecting the native state of casein micelles.

The effect of milk processing on the microstructure of the milk fat globule and rennet induced gel observed using confocal laser scanning microscopy.

Confocal laser scanning microscopy (CLSM) was successfully used to observe the effect of milk processing on the size and the morphology of the milk fat globule in raw milk, raw ultrafiltered milk, and standardized and pasteurized milk prepared for cheese manufacture (cheese-milk) and commercial pasteurized and homogenized milk. Fat globule size distributions for the milk preparations were analyzed using both image analysis and light scattering and both measurements produced similar data trends. Changes to the native milk fat globule membrane (MFGM) were tracked using a MFGM specific fluorescent stain that allowed MFGM proteins and adsorbed proteins to be differentiated on the fat globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the identity of native MFGM proteins isolated from the surface of fat globules within raw, UF retentate, and cheese-milk preparations, whereas only casein was detected on the surface of fat globules in homogenized milk. The microstructure, porosity, and gel strength of the rennet induced gel made from raw milk and cheese-milk was also found to be comparable and significantly different to that made from homogenized milk. Our results highlight the potential use of CLSM as a tool to observe the structural details of the fat globule and associated membrane close to its native environment.

Hot topic: sonication increases the heat stability of whey proteins.

The thickening or gelling of protein-based dairy streams and ingredients upon exposure to heat has been an ongoing problem in dairy processing for many decades. This phenomenon can restrict the range of dairy product options and reduce manufacturing efficiencies by limiting the type and extent of heat treatment that can be used. In this report, we outline a novel approach to overcoming this problem. The use of preheating treatments to induce whey protein aggregate formation in whey products is well known in the field. However, we show that the application of ultrasound for a very short duration after such a heating step breaks down these aggregates and prevents their reformation on subsequent heating, thereby reducing the viscosity increase that is usually associated with this process. This novel technique has the potential to provide significant economic benefit to the dairy manufacturing industry.

Minimising oil droplet size using ultrasonic emulsification.

The efficient production of nanoemulsions, with oil droplet sizes of less than 100nm would facilitate the inclusion of oil soluble bio-active agents into a range of water based foods. Small droplet sizes lead to transparent emulsions so that product appearance is not altered by the addition of an oil phase. In this paper, we demonstrate that it is possible to create remarkably small transparent O/W nanoemulsions with average diameters as low as 40nm from sunflower oil. This is achieved using ultrasound or high shear homogenization and a surfactant/co-surfactant/oil system that is well optimised. The minimum droplet size of 40nm, was only obtained when both droplet deformability (surfactant design) and the applied shear (equipment geometry) were optimal. The time required to achieve the minimum droplet size was also clearly affected by the equipment configuration. Results at atmospheric pressure fitted an expected exponential relationship with the total energy density. However, we found that this relationship changes when an overpressure of up to 400kPa is applied to the sonication vessel, leading to more efficient emulsion production. Oil stability is unaffected by the sonication process.

Ultrasonic enhancement of the supercritical extraction from ginger.

This work examines the concurrent use of power ultrasound during the extraction of pungent compounds from a typical herb (ginger) with supercritical CO(2). A power ultrasonic transducer with an operating frequency of 20 kHz is connected to an extraction vessel and the extraction of gingerols from freeze-dried ginger particles (4-8 mm) is monitored. In the presence of ultrasound, we find that both the extraction rate and the yield increase. The higher extraction rate is attributed to disruption of the cell structures and an increase in the accessibility of the solvent to the internal particle structure, which enhances the intra-particle diffusivity. While cavitation would readily account for such enhancement in ambient processes, the absence of phase boundaries should exclude such phenomena above the critical point. Possible alternate mechanisms for the cell structure damage are discussed.