Sweet taste receptor subunit T1R3 regulates casein secretion and phosphorylation of STAT5 in mammary epithelial cells.

During lactation, mammary epithelial cells (MECs) on the apical membrane are in contact with lactose in milk, while MECs on the basolateral membrane are in contact with glucose in blood. Both glucose and lactose are sweeteners that are sensed by a sweet taste receptor. Previously, we have shown that lactose exposure on the basolateral membrane, but not the apical membrane, inhibits casein production and phosphorylation of STAT5 in MECs. However, it remains unclear whether MECs have a sweet taste receptor. In this study, we confirmed that the sweet taste receptor subunit T1R3 existed in both the apical and basolateral membranes of MECs. Subsequently, we investigated the influence of apical and basolateral sucralose as a ligand for the sweet taste receptor using a cell culture model. In this model, upper and lower media were separated by the MEC layer with less-permeable tight junctions. The results showed in the absence of glucose, both apical and basolateral sucralose induced phosphorylation of STAT5, which is a positive transcriptional factor for milk production. In contrast, the T1R3 inhibitor basolateral lactisole reducing phosphorylated STAT5 and secreted caseins in the presence of glucose. Furthermore, exposure of the apical membrane to sucralose in the presence of glucose inhibited the phosphorylation of STAT5. Simultaneously, GLUT1 was partially translocated from the basolateral membrane to the cytoplasm in MECs. These results suggest that T1R3 functions as a sweet receptor and is closely involved in casein production in MECs.

Enhanced stress resistance of Bifidobacterium breve NRBB57 by induction of stress proteins at near-zero growth rates.

Bifidobacterium breve is a common habitant of the human gut and is used as probiotic in functional foods. B. breve has to cope with multiple stress conditions encountered during processing and passage through the human gut, including high temperature, low pH and exposure to oxygen. Additionally, during industrial processing and in the gut, B. breve could encounter nutrient limitation resulting in reduced growth rates that can trigger adaptive stress responses. For this reason, it is important to develop culture methods that elicit resistance to multiple stresses (robustness) encountered by the bacteria. To investigate the impact of caloric restriction on robustness of the probiotic B. breve NRBB57, this strain was grown in lactose-limited chemostat cultures and in retentostat for 21 days, at growth rates ranging from 0.4 h-1 to 0.00081 h-1. Proteomes of cells harvested at different growth rates were correlated to acid, hydrogen peroxide and heat stress survival capacity. Comparative proteome analysis showed that retentostat-grown cells had significantly increased abundance of a variety of stress proteins involved in protein quality maintenance and DNA repair (DnaJ, Hsp90, FtsH, ClpB, ClpP1, ClpC, GroES, RuvB, RecA), as well as proteins involved in oxidative stress defence (peroxiredoxin, ferredoxin, thioredoxin peroxidase, glutaredoxin and thioredoxin reductase). Exposure to three different stress conditions, 45 °C, pH 3, and 10 mM H2O2, showed highest stress resistance of retentostat cells sampled at week 2 and week 3 grown at 0.0018 and 0.00081 h-1. Our findings show that cultivation at near-zero growth rates induces higher abundance of stress defence proteins contributing to the robustness of B. breve NRBB57, thereby offering an approach that may support its production and functionality.

Development of a dynamic model for prediction of energy in milk protein, lactose, fat, and enteric methane emissions in goats based on energy balance and indirect calorimetry studies.

Feed costs are overwhelmingly the largest expense for dairy producers. Thus, improving milk production efficiency (milk fat and protein are the main incomes for farmers) is of great economic importance in the dairy industry. The main objective of this study was to develop a dynamic energy partitioning model to describe and quantify how dietary energy from carbohydrate, protein, and fat is transferred to milk (protein, lactose, and fat) in dairy goats. In addition, due to increasing worldwide concerns regarding livestock contribution to global warming, methane (CH4) emission was quantified. For modeling purposes, 158 individual goat observations were used and randomly split into 2/3 for model development and 1/3 for internal evaluation. For external evaluation, 20 different energy balance studies from the literature (77 observations) were evaluated. The Root Mean Square Prediction Error (RMSPE) was 13.2% for loss of energy in CH4, 16.8% for energy in fat, 19.4% for energy in protein, and 22.3 energy in lactose. Mean bias was around zero for all variables and the slope bias was zero for milk energy in lactose, close to 1% for milk fat (1.01%), and around 3% and 10% for protein and CH4, respectively. Random bias was greater than 85% for energy in CH4 and milk energy components indicating non-systematic errors and that the equation in the model fitted the data properly. Analyses of residuals appeared to be randomly distributed around zero. Slopes of regression lines for residuals vs. predicted were positive for milk fat energy, zero for lactose, and negative for milk energy in protein and CH4. This model suggested for use with mixed diets and by-products to obtain balanced macronutrient supply, methane emissions, and milk performance during mid lactation could be an interesting tool to help farmers simulate scenarios that increase milk fat and protein, evaluate CH4 emissions, without the costs of running animal trials.

The present model using mixed diets with different by-products to obtain macronutrient balance, methane emission, and milk performance during mid lactation could be an interesting tool to help farmers, without the costs of running animal experiments. The dietary change from grain-based to partial replacement with agro-industrial-byproducts in mid-lactation dairy goats was accompanied by transformations in carbohydrate and fat energy transfer to support production. The output underscored that both oxidation of carbohydrate and fat interact to maintain milk energy output.

Enzymatic Glyco-Modification of Synthetic Membrane Systems.

The present report assesses the capability of a soluble glycosyltransferase to modify glycolipids organized in two synthetic membrane systems that are attractive models to mimic cell membranes: giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs). The objective was to synthesize the Gb3 antigen (Galα1,4Galß1,4Glcß-Cer), a cancer biomarker, at the surface of these membrane models. A soluble form of LgtC that adds a galactose residue from UDP-Gal to lactose-containing acceptors was selected. Although less efficient than with lactose, the ability of LgtC to utilize lactosyl-ceramide as an acceptor was demonstrated on GUVs and SLBs. The reaction was monitored using the B-subunit of Shiga toxin as Gb3-binding lectin. Quartz crystal microbalance with dissipation analysis showed that transient binding of LgtC at the membrane surface was sufficient for a productive conversion of LacCer to Gb3. Molecular dynamics simulations provided structural elements to help rationalize experimental data.

Short-term physiological responses to moderate heat stress in grazing dairy cows in temperate climate.

Even in temperate climate regions, an increase in ambient temperature and exposure to solar radiation can cause heat stress in lactating dairy cows. We hypothesised that grazing dairy cows exhibit short-term physiological changes due to increasing heat load under moderate climate conditions. Over two consecutive summers, 38 lactating Holstein dairy cows were studied in a full-time grazing system. Data were collected in 10 experimental periods of up to three consecutive days with a moderate comprehensive climate index (CCI). The individual animals' vaginal temperature (VT), heart rate, and locomotor activity data were automatically monitored with sensors. Blood samples and proportional whole milk samples were collected at afternoon milking. The concentrations of beta-hydroxybutyrate, glucose, non-esterified fatty acids, urea nitrogen, plasma thyroxine and triiodothyronine were analysed in blood plasma, and fat, protein, lactose, urea nitrogen, cortisol, Na+, K+, and Cl- concentrations were analysed in milk. The daily distribution of VT recordings greater than 39 °C showed a circadian rhythm with a proportion of recordings of 2% and lower during the night and a percentage of 10% or higher in the afternoon. The cows' maximal daily vaginal temperature (VTMAX) between 0830 and 1430 h was positively related to the mean daily CCI in the same time period (CCIMEAN; mean and SD 23.6 ± 5.4 °C). Cows with greater VTMAX had an increased mean heart rate, plasma glucose and milk cortisol concentrations and decreased concentrations of plasma thyroxine and triiodothyronine. The concentration of Na+ in milk was lower, and the concentration of K+ in milk tended to be higher in cows with increased VTMAX. For beta-hydroxybutyrate, non-esterified fatty acids and urea nitrogen concentrations in plasma and fat and lactose concentrations in milk no relationships were found in terms of increasing VT. For milk urea nitrogen and protein concentrations, the proportion of total variance explained by inter-individual or -period variance was high. In conclusion, changes observed in milk and blood likely reflected short-term physiological responses to moderate heat stress. In particular, milk cortisol and Na+ may be useful traits for timely monitoring of heat stress in individual cows because their inter-individual variances were relatively small and samples can be collected non-invasively.

Utilization of delactosed whey permeate for the synthesis of ethyl acetate with Kluyveromyces marxianus.

Ethyl acetate is an important organic solvent and currently produced from fossil carbon resources. Microbial synthesis of this ester from sugar-rich waste could be an interesting alternative. Therefore, synthesis of ethyl acetate by Kluyveromyces marxinanus DSM 5422 from delactosed whey permeate (DWP) was studied in an aerated stirred bioreactor at 40 °C. DWP is mainly composed of residual lactose and minerals. The minerals inhibited yeast growth, as witnessed by an increased lag period, a reduced growth rate, and an extended process duration. All experiments were therefore carried out with diluted DWP. In a series of batch experiments, the pH of iron-deficient DWP medium varied between 4.8 and 5.9. The pH of the cultivation medium significantly influenced cell growth and product syntheses, with the highest ethyl acetate yield of 0.347 g g-1 and lowest by-product formation achieved at pH 5.1. It is likely that this effect is due to pH-dependent iron chelation, which affects the iron bioavailability and the intracellular iron content, thus affecting growth and metabolite synthesis. The viability of yeast cells was always high despite the harsh conditions in DWP medium, which enabled extended usage of the biomass in repeated-batch and fed-batch cultivations. These two culture techniques increased the volume of DWP processed per time by 32 and 84% for the repeated-batch and the fed-batch cultivation, respectively, without a drop of the ester yield. KEY POINTS: • Delactosed whey permeate was converted to ethyl acetate with a high rate and yield. • The formation of ethyl acetate in DWP medium at iron limitation is pH-dependent. • Highly active yeasts from batch processes enabled extension as fed and repeated batch.

Heritabilities and genetic and phenotypic correlations for milk production and fertility traits of spring-calved once-daily or twice-daily milking cows in New Zealand.

The objectives of this study were to estimate the genetic and phenotypic correlations and heritabilities for milk production and fertility traits in spring-calved once-daily (OAD) milking cows for the whole season in New Zealand and compare those estimates with twice-daily (TAD) milking cows. Data used in the study consisted of 69,252 first parity cows from the calving seasons 2015-2016 to 2017-2018 in 113 OAD and 531 TAD milking herds. Heritability estimates for production and fertility traits were obtained through single-trait animal models, and estimates of genetic and phenotypic correlations were obtained through bivariate animal models. Heritability estimates of production traits varied from 0.26 to 0.61 in OAD and from 0.13 to 0.63 in TAD. Heritability estimates for fertility traits were low in both OAD and TAD milking cow populations, and estimates were consistent (OAD: 0.01 to 0.10 and TAD: 0.01 to 0.08) across milking regimens. Estimates of phenotypic and genetic correlations among production traits were consistent across populations. In both populations, phenotypic correlations between milk production and fertility traits were close to zero, and most of the genetic correlations were antagonistic. In OAD milking cows, genetic correlations of milk and lactose yields with the start of mating to conception, 6-wk in-calf, not-in-calf, and 6-wk calving rate were close to zero. Interval from first service to conception was negatively genetically correlated with milk and lactose yields in OAD milking cows. Protein percentage was positively genetically correlated with 3-wk and 6-wk submission, 3-wk in-calf, 6-wk in-calf, first service to conception, 3-wk calving, and 6-wk calving rate in the TAD milking cow population, but these correlations were low in the OAD milking cow population. Further studies are needed to understand the relationship of protein percentage and fertility traits in the OAD milking system. The phenotypic correlations between fertility traits were similar in OAD and TAD milking populations. Genetic correlations between fertility traits were strong (≥0.70) in cows milked TAD, but genetic correlations varied from weak to strong in cows milked OAD. Further research is required to evaluate the interaction between genotype by milking regimen for fertility traits in terms of sire selection in the OAD milking cow population.

Feeding lower-protein diets based on red clover and grass or alfalfa and corn silage does not affect milk production but improves nitrogen use efficiency in dairy cows.

Reducing the dietary crude protein (CP) concentration can decrease the financial cost and lower the environmental impact of milk production. Two studies were conducted to examine the effects of reducing the dietary CP concentration on animal performance, nutrient digestibility, milk fatty acid (FA) profile, and nitrogen use efficiency (NUE; milk N/N intake) in dairy cows fed legume silage-based diets. Thirty-six multiparous Holstein-Friesian dairy cows that were 76 ± 14 (mean ± SD) days in milk and 698 ± 54 kg body weight were used in a 3 × 3 Latin square design in each of 2 studies, with 3 periods of 28 d. In study 1, cows were fed diets based on a 50:50 ratio of red clover to grass silage [dry matter (DM) basis] containing 1 of 3 dietary CP concentrations: high (H) = 175 g of CP/kg of DM; medium (M) = 165 g of CP/kg of DM; or low (L) = 150 g of CP/kg of DM. In study 2, cows were fed 175 g of CP/kg of DM with a 50:50 ratio of alfalfa to corn silage (H50) or 1 of 2 diets containing 150 g of CP/kg of DM with either a 50:50 (L50) or a 60:40 (L60) ratio of alfalfa to corn silage. Cows in both studies were fed a total mixed ration with a forage-to-concentrate ratio of 52:48 (DM basis). All diets were formulated to meet the MP requirements, except L (95% of MP requirements). In study 1, cows fed L ate 1.6 kg of DM/d less than those fed H or M, but milk yield was similar across treatments. Mean milk protein, fat, and lactose concentrations were not affected by diet. However, the apparent total-tract nutrient digestibility was decreased in cows fed L. The NUE was 5.7 percentage units higher in cows fed L than H. Feeding L also decreased milk and plasma urea concentrations by 4.4 mg/dL and 0.78 mmol/L, respectively. We found no effect of dietary treatment on the milk saturated or monounsaturated FA proportion, but the proportion of polyunsaturated FA was increased, and milk odd- and branched-chain FA decreased in cows fed L compared with H. In study 2, DM intake was 2 kg/d lower in cows receiving L50 than H50. Increasing the alfalfa content and feeding a low-CP diet (L60) did not alter DMI but decreased milk yield and milk protein concentration by 2 kg/d and 0.6 g/kg, respectively, compared with H50. Likewise, milk protein and lactose yield were decreased by 0.08 kg/d in cows receiving L60 versus H50. Diet had no effect on apparent nutrient digestibility. Feeding the low-CP diets compared with H50 increased the apparent NUE by approximately 5 percentage units and decreased milk and plasma urea concentrations by 7.2 mg/dL and 1.43 mmol/L, respectively. Dietary treatment did not alter milk FA profile except cis-9,trans-11 conjugated linoleic acid, which was higher in milk from cows receiving L60 compared with H50. We concluded that reducing CP concentration to around 150 g/kg of DM in red clover and grass or alfalfa and corn silage-based diets increases the apparent NUE and has little effect on nutrient digestibility or milk performance in dairy cows.

Galactomyces candidus diversity in the complex mycobiota of cow-milk bryndza cheese comprising antagonistic and sensitive strains.

Galactomyces candidus (orthographic variant: Galactomyces candidum) is a heterogeneous species of Saccharomycetales that comprises dimorphic yeasts described previously under various names (e.g. Geotrichum, Dipodascus). Its strains are common components of the cheese surface mycobiota. This study identified genetically and physiologically heterogeneous G. candidus strains in the complex mycobiota of artisanal cow-milk bryndza samples. The traditional Slovak bryndza is a cheese produced from ewe's milk in cooler mountainous regions and from cow's milk in warmer low-land regions. The taxonomic analysis of the culturable yeasts of the latter version carried out in this study revealed considerable differences from the yeast biota previously described for ovine bryndza. However, the conventional D1/D2- and ITS-based barcode analyses could not assign unanimously all isolates to species because of the intragenomic barcode diversity in certain groups and the discordance between the D1/D2 and ITS results in other groups. The identified species and groups of isolates had different abilities to utilise the carbon and energy sources (lactose, lactate, lipids and proteins) available in milk and ripening cheese. The G. candidus strains did not metabolise lactose and lactate, hydrolysed milk proteins with diverse, usually moderate efficiency and only could grow on certain amino acids as only energy sources. Their preferred substrate was lipid. Under aerobic conditions, its hyphae penetrated the lipid droplets and degraded their content from inside by developing a dense internal mycelium. Sporulation and different MLST (multilocus sequence typing) patterns indicated that the Galactomyces strains could sexually interact and their genomes could recombine. The Galactomyces and Kluyveromyces isolates had antagonistic effects against other members of the mycobiota.

Biosynthesis of Human Milk Oligosaccharides: Enzyme Cascade and Metabolic Engineering Approaches.

Human milk oligosaccharides (HMOs) have unique beneficial effects for infants and are considered as the new gold standard for premium infant formula. They are a collection of unconjugated glycans, and more than 200 distinct structures have been identified. Generally, HMOs are enzymatically produced by elongation and/or modification from lactose via stepwise glycosylation. Each glycosylation requires a specific glycosyltransferase (GT) and the corresponding nucleotide sugar donor. In this review, the typical HMO-producing GTs and the one-pot multienzyme modules for generating various nucleotide sugar donors are introduced, the principles for designing the enzyme cascade routes for HMO synthesis are described, and the important metabolic engineering strategies for mass production of HMOs are also reviewed. In addition, the future research directions in biotechnological production of HMOs were prospected.

Live yeast supplementation altered the bacterial community's composition and function in rumen and hindgut and alleviated the detrimental effects of heat stress on dairy cows.

The objective of this study was to investigate the effects of live yeast (LY, Saccharomyces cerevisiae) on the lactation performance, bacterial community, and functions in the rumen and hindgut of dairy cows under heat stress. Thirty-three multiparous (parity 3.9 ± 0.8) Holstein dairy cows (189.1 ± 6.6 d in milk at the beginning of the experiment) were randomly assigned to three groups (11 cows per treatment). Cows in the three groups were fed a diet without yeast (CON), with 10 g yeast/d/head (LY-10), and with 20 g yeast/d/head (LY-20). The yeast product contained 2.0 × 1010 CFU/g. Supplementing LY decreased the rectal temperature and respiratory rate of cows, and increased dry matter intake, milk yield, milk fat yield, milk protein yield, and milk lactose yield (P < 0.001), yet decreased milk urea nitrogen concentration (P = 0.035). Interaction effects of treatment × week were observed for rectal temperature (P < 0.05), respiratory rate (P < 0.05), milk yield (P = 0.015), milk urea nitrogen (P = 0.001), milk protein yield (P = 0.008), and milk lactose yield (P = 0.030). In rumen, LY increased the concentrations of acetate, isobutyrate, isovaterate, valerate, total volatile fatty acids (VFAs), and NH3-N (P < 0.05). Miseq sequencing of the 16S rRNA genes showed that LY increased the relative abundance of Prevotella and Prevotellaceae UCG-003 at the genus level with a series of enriched pathways in the metabolism of carbohydrates and protein. In fecal samples, LY did not affect the profile of VFAs (P > 0.05). Clostridium sensu stricto 1 (P = 0.013) and Actinobacillus (P = 0.011) increased in the relative abundance by LY, whereas Bacteroides (P = 0.016) and Oscillospirales UCG-010 (P = 0.005) decreased with a series of enriched pathways in carbohydrate metabolism, secondary bile acid biosynthesis. In summary, LY supplementation altered the bacterial community's composition and function in rumen and hindgut, and simultaneously alleviated the detrimental effects of heat stress on dairy cows. These findings provide extended insight into the effects of LY in the rumen and hindgut of dairy cows exposed to heat stress.

Dairy cows are exposed to severe heat stress under hot and humid climates in summer in south China, resulting in a decline in feed intake and milk yield. Therefore, we investigated the effect of live yeast (LY, Saccharomyces cerevisiae) supplementation on the milk performance, bacterial community, and functions in the rumen and hindgut of dairy cows under heat stress. Thirty-three dairy cows were randomly assigned to control (CON, without yeast addition), treatment 1 (LY-10, with 10 g yeast/d/head) and treatment 2 (LY-20, with 20 g yeast/d/head). Supplementing LY decreased the rectal temperature and respiratory rate of the dairy cows and increased feed intake and milk performance. Live yeast enhanced fermentation in the rumen but did not affect it in the hindgut. Live yeast altered the microbiota in the rumen and hindgut, with an enrichment of bacteria in the pathways of the metabolism of carbohydrates, protein, and other substances. In all, LY supplementation had beneficial effects on dairy cows under heat stress by affecting the microbiota and fermentation in the rumen and hindgut.

Biosynthesis of lactosucrose by a new source of ß-fructofuranosidase from Bacillus methanolicus LB-1.

Lactosucrose (LS) is a prebiotic trisaccharide enzymatically synthesized by transglycosylation from lactose and sucrose with beneficial health effect. The ß-fructofuranosidase used for synthesis of LS was produced from Bacillus methanolicus LB-1, which was isolated from traditional rice wine. A maximal yield of 8.63 U/mL of the enzyme was obtained by fermentation with B. methanolicus LB-1 under the optimized conditions: 10 g/L of glucose, 5 g/L of yeast extract, initial medium pH at 7.0, 37 °C, 24 h. The enzyme was purified and identified by ammonium sulfate fractional precipitation, Sephadex G-75 gel filtration chromatography and LC-MS, and SDS-PAGE of the purified enzyme showed a major protein band at 45 kDa. Biosynthesis of LS was performed using the purified ß-fructofuranosidase, and production of LS reached 110 g/L under the optimized reaction conditions: pH at 7.0, 37 °C, 6.0 U/g sucrose of enzyme, 15% of sucrose, 15% of lactose, 28 h. HPLC analysis of the reaction products showed a distinct peak for LS at about 30 min of elution, confirming that B. methanolicus LB-1 ß-fructofuranosidase had effective transfructosylation activity. Therefore, this new microbial source of ß-fructofuranosidase may be a candidate with potential application prospect in biosynthesis of prebiotic LS.

Increasing Lovastatin Production by Re-routing the Precursors Flow of Aspergillus terreus via Metabolic Engineering.

Lovastatin is an anti-cholesterol medicine that is commonly prescribed to manage cholesterol levels, and minimise the risk of suffering from heart-related diseases. Aspergillus terreus (ATCC 20542) supplied with carbohydrates or sugar alcohols can produce lovastatin. The present work explored the application of metabolic engineering in A. terreus to re-route the precursor flow towards the lovastatin biosynthetic pathway by simultaneously overexpressing the gene for acetyl-CoA carboxylase (acc) to increase the precursor flux, and eliminate ( +)-geodin biosynthesis (a competing secondary metabolite) by removing the gene for emodin anthrone polyketide synthase (gedC). Alterations to metabolic flux in the double mutant (gedCΔ*accox) strain and the effects of using two different substrate formulations were examined. The gedCΔ*accox strain, when cultivated with a mixture of glycerol and lactose, significantly (p < 0.05) increased the levels of metabolic precursors malonyl-CoA (48%) and acetyl-CoA (420%), completely inhibited the (+)-geodin biosynthesis, and increased the level of lovastatin [152 mg/L; 143% higher than the wild-type (WT) strain]. The present work demonstrated how the manipulation of A. terreus metabolic pathways could increase the efficiency of carbon flux towards lovastatin, thus elevating its overall production and enabling the use of glycerol as a substrate source. As such, the present work also provides a framework model for other medically or industrially important fungi to synthesise valuable compounds using sustainable carbon sources.

Casein Lactose-Glycation of the Maillard-Type Attenuates the Anti-Inflammatory Potential of Casein Hydrolysate to IEC-6 Cells with Lipopolysaccharide Stimulation.

During the thermal processing of dairy products, the Maillard reaction occurs between milk proteins and lactose, resulting in the formation of various products including glycated proteins. In this study, lactose-glycated casein was generated through the Maillard reaction between casein and lactose and then hydrolyzed by a trypsin preparation. The anti-inflammatory effect of the resultant glycated casein hydrolysate (GCH) was investigated using the lipopolysaccharide (LPS)-sitmulated rat intestinal epithelial (IEC-6) cells as a cell model and corresponding casein hydrolysate (CH) as a control. The results indicated that the preformed glycation enabled lactose conjugation to casein, which endowed GCH with a lactose content of 12.61 g/kg protein together with a lower activity than CH to enhance the viability value of the IEC-6 cells. The cells with LPS stimulation showed significant inflammatory responses, while a pre-treatment of the cells with GCH before LPS stimulation consistently led to a decreased secretion of three pro-inflammatory mediators, namely, IL-6, IL-1ß and tumor necrosis factor-α (TNF-α) but an increased secretion of two anti-inflammatory mediators, including IL-10 and transforming growth factor-ß (TGF-ß), demonstrating the anti-inflammatory potential of GCH in LPS-stimulated cells. In addition, GCH up-regulated the expression of TLR4, p-p38, and p-p65 proteins in the stimulated cells, resulting in the suppression of NF-κB and MAPK signaling pathways. Collectively, GCH was mostly less efficient than CH to exert these assessed anti-inflammatory activities in the cells and more importantly, GCH also showed an ability to cause cell inflammation by promoting IL-6 secretion and up-regulating the expression of TLR4 and p-p65. The casein lactose-glycation of the Maillard-type was thereby concluded to attenuate the anti-inflammatory potential of the resultant casein hydrolysate. It is highlighted that the casein lactose-glycation of the Maillard-type might cause a negative impact on the bioactivity of casein in the intestine, because the glycated casein after digestion could release GCH with reduced anti-inflammatory activity.

Module-Guided Metabolic Rewiring for Fucosyllactose Biosynthesis in Engineered Escherichia coli with Lactose De Novo Pathway.

Fucosyllactose (FL) has garnered considerable attention for its benefits on infant health. In this study, we report an efficient E. coli cell factory to produce 2'/3-fucosyllactose (2'/3-FL) with lactose de novo pathway through metabolic network remodeling, including (1) modification of the PTSGlc system to enhance glucose internalization efficiency; (2) screening for ß-1,4-galactosyltransferase (ß-1,4-GalT) and introduction of lactose synthesis pathway; (3) eliminating inhibition of byproduct pathways; (4) constructing antibiotic-free and inducer-free FL strains; and (5) up-regulating the expression of genes in the GDP-l-fucose module. The final engineered strains BP10-3 and BP11-3 produced 4.36 g/L for 2'-FL and 3.23 g/L for 3-FL in shake flasks. In 3 L bioreactors, fed-batch cultivations of the two strains produced 40.44 g/L for 2'-FL and 30.42 g/L for 3-FL, yielding 0.63 and 0.69 g/g glucose, respectively. The strategy described in this work will help to engineer E. coli as a safe chassis for other lactose-independent HMOs production.

Bayesian evaluation of the effect of non-genetic factors on the phenomics for quality-related milk nutrients and yield in Murciano-Granadina goats.

The aim of this study was to evaluate the effect of non-genetic factors on the variability of milk production and composition using Bayesian linear regression. We analyzed 2594 milk records from 159 dairy goats from the breeding nucleus of the Murciano-Granadina breed. Bayesian linear regression was used to determine the effects of non-genetic factors on the phenomics for quality-related milk nutrients and yield. Multivariate regression model significantly explained 21.5%, 40.0%, 41.5%, 44.3%, 44.6%, and 47.5% of the variability in somatic cell count (SCC, sc/mL), lactose (%), protein (%), milk yield (kg), fat (%), and dry matter (%), respectively. Although the aforementioned factor combination significantly conditions milk production and composition, SCC may be particularly affected by collateral factors. Milking routine and drying period factors are reference predictors to be considered in the evaluation of milk production and composition progression. Drying period extensions positively repercussed on milk yield and lactose content, but negatively affected fat, protein, dry matter contents, and somatic cell count. Variability across drying years may depend on the drying season rather than the drying month course, except for milk yield, for which an increasing trend was reported from winter to summer. Including drying period-related non-genetic factors in genetic evaluations improves the accuracy of the regression models and permits to boost the commercial possibilities and profitability of local breeds.

Recent innovations in immobilization of ß-galactosidases for industrial and therapeutic applications.

Immobilized enzymes are better suited for industrial applications than free enzymes due to their favorable properties such as ease of separation and reuse, and enhanced stability and storage life. ß-Galactosidases are an important class of glycosidases with hydrolysis and transglycosylation activities, which are applied in industries for lactose hydrolysis and prebiotics synthesis worldwide. The recent innovations in immobilized ß-galactosidases have improved the performance of the immobilized enzymes and broadened their applications in the fields of food, energy, and medicine. Innovations in ß-galactosidase immobilization methods include rational adsorption based on enzyme features, layer by layer adsorption for strengthened ionic bonding, 3-D printing for rapid and elaborate entrapment, modifications of either materials or enzymes for ingenious covalent binding, nontoxic crosslinking, carrier-free immobilization, and oriented immobilization either through protein engineering or enzyme display on cells, membranes, and phages, along with innovations in carrier materials involving the introduction of graphene derivatives, polyaniline nanomaterials, nanofibers, nucleotide molecules, Langmuir-Blodgett films, and so on. These innovations have partially solved the problems associated with traditional methods, resulting in enzymes with highly retained activity, excellent stability, reduced microbial contamination, enzyme leakage, and reagent toxicity. The immobilized ß-galactosidases with potential economic and environmental benefits have been extendedly used for hydrolysis of prodrugs for disease treatment, assembly of biosensors for lactose detection, synthesis of bioactive carbohydrates, and even production of food additives and industrial products, such as tagatose and bioethanol. This review describes the innovations in ß-galactosidases immobilization and the applications of these immobilized enzymes. It not only enables the fully understanding of ß-galactosidases, but also provides a valuable reference for the immobilization of other industrially-important enzymes.

Enhanced thermal stability of the ß-galactosidase BgaB from Bacillus circulans by cyclization mediated via SpyTag/SpyCatcher interaction and its use in galacto-oligosaccharides synthesis.

Cyclization of proteins using SpyTag/SpyCatcher is a novel approach to increase their thermal stability. In this paper, we test this approach on two ß-galactosidases from Bacillus circulans, BgaB and BgaC, and find that BgaB was stabilized while BgaC was not. Wild-type BgaB precipitated completely upon heating above 70 °C, but after SpyRing cyclization, it remained soluble after heating to 90 °C. Similarly, wild-type BgaB retained only 50 % activity after heating at 60 °C for 10 min, but this increased to 80 % after SpyRing cyclization. In contrast, cyclization decreased the stability of BgaC. After SpyRing cyclization, BgaC only retained 2 % activity after 20-min incubation at 55 °C, whereas the wild-type BgaC retained 25 % activity. One reason for the different effect of cyclization may the shorter distance between the N- and C-termini in BgaB (20.2 Å) as compared to BgaC (43.7 Å). The intrinsic fluorescence and circular dichroism spectra suggested that SpyRing cyclization of BgaB did not significantly change its conformation or secondary structure. SpyRing cyclized BgaB yielded similar amounts and compositions of galacto-oligosaccharides using a high initial lactose concentration (40 %, w/v), but a slightly higher amount at low initial lactose concentration (5 %, w/v) suggesting increased transgalactosylation activity.

Strategies for Enhancing Microbial Production of 2'-Fucosyllactose, the Most Abundant Human Milk Oligosaccharide.

Human milk oligosaccharides (HMOs), a group of structurally diverse unconjugated glycans in breast milk, act as important prebiotics and have plenty of unique health effects for growing infants. 2'-Fucosyllactose (2'-FL) is the most abundant HMO, accounting for approximately 30%, among approximately 200 identified HMOs with different structures. 2'-FL can be enzymatically produced by α1,2-fucosyltransferase, using GDP-l-fucose as donor and lactose as acceptor. Metabolic engineering strategies have been widely used for enhancement of GDP-l-fucose supply and microbial production of 2'-FL with high productivity. GDP-l-fucose supply can be enhanced by two main pathways, including de novo and salvage pathways. 2'-FL-producing α1,2-fucosyltransferases have widely been identified from various microorganisms. Metabolic pathways for 2'-FL synthesis can be basically constructed by enhancing GDP-l-fucose supply and introducing α1,2-fucosyltransferase. Various strategies have been attempted to enhance 2'-FL production, such as acceptor enhancement, donor enhancement, and improvement of the functional expression of α1,2-fucosyltransferase. In this review, current progress in GDP-l-fucose synthesis and bacterial α1,2-fucosyltransferases is described in detail, various metabolic engineering strategies for enhancing 2'-FL production are comprehensively reviewed, and future research focuses in biotechnological production of 2'-FL are suggested.

Identification, kinetics and thermodynamic analysis of novel ß-galactosidase from Convolvulus arvensis seeds: An efficient agent for delactosed milk activity.

The ß-galactosidase was extracted and purified from 100 g of C. arvensis seeds using a variety of protein purification procedures such as ammonium sulphate fractionation, gel filtration, and finally chromatography on a cationic ion exchanger. The effects of metal ions, kinetics parameters, and glycoprotein nature were determined, as well as the optimal pH and temperature of the purified enzyme. With a high specific activity (72 units/mg), ß-galactosidase was isolated to a 24-fold apparent electrophoretic homogeneity. The molecular mass of ß-galactosidase was determined as monomeric, which was further confirmed by SDS-PAGE and MALDI-TOF/MS analysis, with a 45 kDa molecular weight. The enzyme has a Km of 0.33 mM and a Vmax of 42 µmol/min Lactose in milk was reduced by 38.5 and 70 % after 4 h of incubation with ß-galactosidase from C. arvensis. The ß-galactosidase thermal inactivation kinetic parameters ΔH°, ΔS°, and ΔG° were calculated, indicating that the enzyme undergoes significant unfolding events during denaturation. Using ß-galactosidase from C. arvensis seeds, lactose hydrolysis in milk up to approx. 50 % was observed. The findings indicate the potential use of C. arvensis seeds for the production of low/delactosed milk for lactose-intolerant population.